secondary antibody from immunostainigs

[Shuchi Gupta]

hello,
  i want to know if somebody knows any good secondary antibodies(  
alexa, atto conjugated) against rabbit which i can use for the  
immunostainings. i have tried  antibodies from cell signaling, abnova  
and dako.but none of them give me the specific signal .( this i get to  
know from my controls which contain only my secondary antibody) .  i  
am using these antibodies in mouse platelets.i block them with BSA 2%  
after fixing and permeabilization.
hope to get the suggestions soon.
thank you
shuchi

[Dries Vercauteren]

Dear Shuchi,

did you try goat anti rabbit-Alexa Fluor 594 (Mol prob; A11012) yet?

No commercial interest.

Good luck,

Dries

2009/3/9 Shuchi Gupta <[hidden email]>

hello,
 i want to know if somebody knows any good secondary antibodies( alexa, atto conjugated) against rabbit which i can use for the immunostainings. i have tried  antibodies from cell signaling, abnova and dako.but none of them give me the specific signal .( this i get to know from my controls which contain only my secondary antibody) .  i am using these antibodies in mouse platelets.i block them with BSA 2% after fixing and permeabilization.
hope to get the suggestions soon.
thank you
shuchi

--
Dries Vercauteren, PhD student
Master of Bioscience Engineering: Cell and Gene Biotechnology

Ghent Research Group on Nanomedicines
www.ugent.be/fw/en/research/biofys
Faculty of pharmaceutical sciences, Ghent University
Harelbekestraat 72, 9000 Ghent
Belgium

Phone:  [phone]
Mobile: [phone]
E-mail: [hidden email]
          [hidden email]

[Bruno Saubaméa]

Dear Shuchu,

what do you think is the problem :the bg is low but you don't have a specific signal or the bg is so high that the specific signal can't be seen?

bruno

 

Bruno SAUBAMEA

 

EA 3621 & Service Commun d'Imagerie Cellulaire et Moléculaire

 

Faculté des Sciences Pharmaceutiques et Biologiques
Université Paris Descartes
4, avenue de l'Observatoire
75006 PARIS

 

tel : [phone]
fax : [phone]

----- Original Message -----

From: [hidden email]

To: [hidden email]

Sent: Monday, March 09, 2009 5:23 PM

Subject: secondary antibody from immunostainigs

hello,
  i want to know if somebody knows any good secondary antibodies( 
alexa, atto conjugated) against rabbit which i can use for the 
immunostainings. i have tried  antibodies from cell signaling, abnova 
and dako.but none of them give me the specific signal .( this i get to 
know from my controls which contain only my secondary antibody) .  i 
am using these antibodies in mouse platelets.i block them with BSA 2% 
after fixing and permeabilization.
hope to get the suggestions soon.
thank you
shuchi

[mmodel]

It seems to me the problem most likely lies with either primary antibody or sample preparation

 

From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Dries Vercauteren
Sent: Monday, March 09, 2009 12:45 PM
To: [hidden email]
Subject: Re: secondary antibody from immunostainigs

 

Dear Shuchi,

did you try goat anti rabbit-Alexa Fluor 594 (Mol prob; A11012) yet?

No commercial interest.

Good luck,

Dries

2009/3/9 Shuchi Gupta <[hidden email]>

hello,
 i want to know if somebody knows any good secondary antibodies( alexa, atto conjugated) against rabbit which i can use for the immunostainings. i have tried  antibodies from cell signaling, abnova and dako.but none of them give me the specific signal .( this i get to know from my controls which contain only my secondary antibody) .  i am using these antibodies in mouse platelets.i block them with BSA 2% after fixing and permeabilization.
hope to get the suggestions soon.
thank you
shuchi

--
Dries Vercauteren, PhD student
Master of Bioscience Engineering: Cell and Gene Biotechnology

Ghent Research Group on Nanomedicines
www.ugent.be/fw/en/research/biofys
Faculty of pharmaceutical sciences, Ghent University
Harelbekestraat 72, 9000 Ghent
Belgium

Phone:  [phone]
Mobile: [phone]
E-mail: [hidden email]
          [hidden email]

[js1719]

Are you getting nonspecific background in the nucleus? We often find that the Molecular Probes Alexa in 568 and 594 have significant background labeling of the nuclear region- not sure why. It can be reduced by storing your secondary in aliquoits in the -80C to avoid multiple freeze thaws and to spin the tube at 13K for 10 minutes prior to use. 

good luck

Julia

On Mar 9, 2009, at 1:34 PM, MODEL, MICHAEL wrote:

It seems to me the problem most likely lies with either primary antibody or sample preparation

 

From: Confocal Microscopy List [[hidden email]] On Behalf Of Dries Vercauteren
Sent: Monday, March 09, 2009 12:45 PM
To: [hidden email]
Subject: Re: secondary antibody from immunostainigs

 

Dear Shuchi,

did you try goat anti rabbit-Alexa Fluor 594 (Mol prob; A11012) yet?

No commercial interest.

Good luck,

Dries

2009/3/9 Shuchi Gupta <[hidden email]>

hello,
 i want to know if somebody knows any good secondary antibodies( alexa, atto conjugated) against rabbit which i can use for the immunostainings. i have tried  antibodies from cell signaling, abnova and dako.but none of them give me the specific signal .( this i get to know from my controls which contain only my secondary antibody) .  i am using these antibodies in mouse platelets.i block them with BSA 2% after fixing and permeabilization.
hope to get the suggestions soon.
thank you
shuchi

-- 
Dries Vercauteren, PhD student
Master of Bioscience Engineering: Cell and Gene Biotechnology

Ghent Research Group on Nanomedicines
www.ugent.be/fw/en/research/biofys
Faculty of pharmaceutical sciences, Ghent University
Harelbekestraat 72, 9000 Ghent
Belgium

Phone:  [phone]
Mobile: [phone]
E-mail: [hidden email]
          [hidden email]

[Ignatius, Mike]

We sell a product, Image-iT FX Signal Enhancer, that was designed to alleviate this type of non-specific binding.  It works especially well with background labeling of the nucleus (and myelin) that can occur with many dyes including the Alexa Fluor line.  The product information sheet or PIS available on the web for this product details what dyes it works best with, and includes the aforementioned 568 and 594.

 

Long name, but short protocol - 30 mins at RT.  Use it in addition to protein blockers and the like, though some papers report seeing it work without additional protein blocking steps - just depends on the source of non-specific signal. 

 

For non-specific, background labeling with Alexa Fluor labeled secondaries, the noise can be reduced by as much as 90%.  For rare event detection and thus long signal integration times it can be very helpful.

 

Mike Ignatius

 

Molecular Probes/Life Technology

---

From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of js1719
Sent: Monday, March 09, 2009 11:14 AM
To: [hidden email]
Subject: Re: secondary antibody from immunostainigs

Are you getting nonspecific background in the nucleus? We often find that the Molecular Probes Alexa in 568 and 594 have significant background labeling of the nuclear region- not sure why. It can be reduced by storing your secondary in aliquoits in the -80C to avoid multiple freeze thaws and to spin the tube at 13K for 10 minutes prior to use. 

good luck

Julia

On Mar 9, 2009, at 1:34 PM, MODEL, MICHAEL wrote:

It seems to me the problem most likely lies with either primary antibody or sample preparation

From: Confocal Microscopy List [[hidden email]] On Behalf Of Dries Vercauteren
Sent: Monday, March 09, 2009 12:45 PM
To: [hidden email]
Subject: Re: secondary antibody from immunostainigs

Dear Shuchi,

did you try goat anti rabbit-Alexa Fluor 594 (Mol prob; A11012) yet?

No commercial interest.

Good luck,

Dries

2009/3/9 Shuchi Gupta <[hidden email]>

hello,
 i want to know if somebody knows any good secondary antibodies( alexa, atto conjugated) against rabbit which i can use for the immunostainings. i have tried  antibodies from cell signaling, abnova and dako.but none of them give me the specific signal .( this i get to know from my controls which contain only my secondary antibody) .  i am using these antibodies in mouse platelets.i block them with BSA 2% after fixing and permeabilization.
hope to get the suggestions soon.
thank you
shuchi

-- 
Dries Vercauteren, PhD student
Master of Bioscience Engineering: Cell and Gene Biotechnology

Ghent Research Group on Nanomedicines
www.ugent.be/fw/en/research/biofys
Faculty of pharmaceutical sciences, Ghent University
Harelbekestraat 72, 9000 Ghent
Belgium

Phone:  [phone]
Mobile: [phone]
E-mail: [hidden email]
          [hidden email]

[Shuchi Gupta]

hello Bruno SAUBAMEA
what i could see from my stainings, it seems that the background is so  
high that the specific signal canot be seen. to get rid of the high  
background i tried washing with high salt PBS but it seems to have  
done no improvement to my samples.
  thank you
  Shuchi Gupta
Rudolf Virchow Centre for experimental medicine.
Uni Wuerzburg
Germany




Quoting Bruno Saubaméa <[hidden email]>:

> Dear Shuchu,
> what do you think is the problem :the bg is low but you don't have a  
> specific signal or the bg is so high that the specific signal can't  
> be seen?
> bruno
>
> Bruno SAUBAMEA
>
> EA 3621 & Service Commun d'Imagerie Cellulaire et Moléculaire
>
> Faculté des Sciences Pharmaceutiques et Biologiques
> Université Paris Descartes
> 4, avenue de l'Observatoire
> 75006 PARIS
>
> tel : [phone]
> fax : [phone]
>   ----- Original Message -----
>   From: Shuchi Gupta
>   To: [hidden email]
>   Sent: Monday, March 09, 2009 5:23 PM
>   Subject: secondary antibody from immunostainigs
>
>
>   hello,
>     i want to know if somebody knows any good secondary antibodies(
>   alexa, atto conjugated) against rabbit which i can use for the
>   immunostainings. i have tried  antibodies from cell signaling, abnova
>   and dako.but none of them give me the specific signal .( this i get to
>   know from my controls which contain only my secondary antibody) .  i
>   am using these antibodies in mouse platelets.i block them with BSA 2%
>   after fixing and permeabilization.
>   hope to get the suggestions soon.
>   thank you
>   shuchi
>

[Bruno Saubaméa]

If the problem is indeed high non specific signal I would recommend blocking with PBS, BSA 1%, Goat serum 5 to 10% if your secondary antibody is made in goat. I routinely use highly cross adsorbed goat anti rabbit coupled to alexa fluor (Invitrogen) diluted 1:200 for immunostaining mouse tissue and get no NS signal.

If binding by Fc receptor can be a problem (I know nothing about receptors in platelets membrane), you can use Fab'2 fragment of the above secondary antibodies.

Hope this can help

bruno

 

Bruno SAUBAMEA

 

EA 3621 & Service Commun d'Imagerie Cellulaire et Moléculaire

 

Faculté des Sciences Pharmaceutiques et Biologiques
Université Paris Descartes
4, avenue de l'Observatoire
75006 PARIS

 

tel : [phone]
fax : [phone]

----- Original Message -----

From: [hidden email]

To: [hidden email]

Sent: Tuesday, March 10, 2009 10:32 AM

Subject: Re: secondary antibody from immunostainigs

hello Bruno SAUBAMEA
what i could see from my stainings, it seems that the background is so 
high that the specific signal canot be seen. to get rid of the high 
background i tried washing with high salt PBS but it seems to have 
done no improvement to my samples.
  thank you
  Shuchi Gupta
Rudolf Virchow Centre for experimental medicine.
Uni Wuerzburg
Germany




Quoting Bruno Saubaméa <[hidden email]>:

> Dear Shuchu,
> what do you think is the problem :the bg is low but you don't have a 
> specific signal or the bg is so high that the specific signal can't 
> be seen?
> bruno
>
> Bruno SAUBAMEA
>
> EA 3621 & Service Commun d'Imagerie Cellulaire et Moléculaire
>
> Faculté des Sciences Pharmaceutiques et Biologiques
> Université Paris Descartes
> 4, avenue de l'Observatoire
> 75006 PARIS
>
> tel : [phone]
> fax : [phone]
>   ----- Original Message -----
>   From: Shuchi Gupta
>   To: [hidden email]
>   Sent: Monday, March 09, 2009 5:23 PM
>   Subject: secondary antibody from immunostainigs
>
>
>   hello,
>     i want to know if somebody knows any good secondary antibodies(
>   alexa, atto conjugated) against rabbit which i can use for the
>   immunostainings. i have tried  antibodies from cell signaling, abnova
>   and dako.but none of them give me the specific signal .( this i get to
>   know from my controls which contain only my secondary antibody) .  i
>   am using these antibodies in mouse platelets.i block them with BSA 2%
>   after fixing and permeabilization.
>   hope to get the suggestions soon.
>   thank you
>   shuchi
>

[anh2006]

Are you staining mouse tissue sections?
If so, is your secondary antibody cross adsorbed
against mouse so that you won't get cross
reactivity from the IgG in the blood?

Jackson ImmunoResearch sells secondary antibodies
conjugated to a variety of fluorophores which are
excellent for staining animal tissues with
polyclonal antibodies. www.jacksonimmuno.com. I
normally don't buy secondaries from any other
source.

>hello Bruno SAUBAMEA
>what i could see from my stainings, it seems
>that the background is so high that the specific
>signal canot be seen. to get rid of the high
>background i tried washing with high salt PBS
>but it seems to have done no improvement to my
>samples.
>  thank you
>  Shuchi Gupta
>Rudolf Virchow Centre for experimental medicine.
>Uni Wuerzburg
>Germany
>
>
>
>
>Quoting Bruno Saubaméa <[hidden email]>:
>
>>Dear Shuchu,
>>what do you think is the problem :the bg is low
>>but you don't have a specific signal or the bg
>>is so high that the specific signal can't be
>>seen?
>>bruno
>>
>>Bruno SAUBAMEA
>>
>>EA 3621 & Service Commun d'Imagerie Cellulaire et Moléculaire
>>
>>Faculté des Sciences Pharmaceutiques et Biologiques
>>Université Paris Descartes
>>4, avenue de l'Observatoire
>>75006 PARIS
>>
>>tel : [phone]
>>fax : [phone]
>>   ----- Original Message -----
>>   From: Shuchi Gupta
>>   To: [hidden email]
>>   Sent: Monday, March 09, 2009 5:23 PM
>>   Subject: secondary antibody from immunostainigs
>>
>>
>>   hello,
>>     i want to know if somebody knows any good secondary antibodies(
>>   alexa, atto conjugated) against rabbit which i can use for the
>>   immunostainings. i have tried  antibodies from cell signaling, abnova
>>   and dako.but none of them give me the specific signal .( this i get to
>>   know from my controls which contain only my secondary antibody) .  i
>>   am using these antibodies in mouse platelets.i block them with BSA 2%
>>   after fixing and permeabilization.
>>   hope to get the suggestions soon.
>>   thank you
>>   shuchi

--

[Glen MacDonald-2]

Dear Shuchi,
I don't believe that you ever provided your current dilutions and  
incubation times.  We commonly use the Molecular Probes Alexa  
conjugated secondaries at 1/200 to 1/500, depending upon incubation  
time for frozen, paraffin, vibratome sections of brain and whole  
cochlea or vestibular organs.  have you performed a dilution series  
with secondary antibody alone for testing background?   Are you making  
certain that the sections are not allowed to dry out?

Regards,
Glen
On Mar 10, 2009, at 5:22 AM, Bruno Saubaméa wrote:

> If the problem is indeed high non specific signal I would recommend  
> blocking with PBS, BSA 1%, Goat serum 5 to 10% if your secondary  
> antibody is made in goat. I routinely use highly cross adsorbed goat  
> anti rabbit coupled to alexa fluor (Invitrogen) diluted 1:200 for  
> immunostaining mouse tissue and get no NS signal.
> If binding by Fc receptor can be a problem (I know nothing about  
> receptors in platelets membrane), you can use Fab'2 fragment of the  
> above secondary antibodies.
> Hope this can help
> bruno
>
> Bruno SAUBAMEA
>
> EA 3621 & Service Commun d'Imagerie Cellulaire et Moléculaire
>
> Faculté des Sciences Pharmaceutiques et Biologiques
> Université Paris Descartes
> 4, avenue de l'Observatoire
> 75006 PARIS
>
> tel : [phone]
> fax : [phone]
> ----- Original Message -----
> From: Shuchi Gupta
> To: [hidden email]
> Sent: Tuesday, March 10, 2009 10:32 AM
> Subject: Re: secondary antibody from immunostainigs
>
> hello Bruno SAUBAMEA
> what i could see from my stainings, it seems that the background is so
> high that the specific signal canot be seen. to get rid of the high
> background i tried washing with high salt PBS but it seems to have
> done no improvement to my samples.
>   thank you
>   Shuchi Gupta
> Rudolf Virchow Centre for experimental medicine.
> Uni Wuerzburg
> Germany
>
>
>
>
> Quoting Bruno Saubaméa <[hidden email]>:
>
> > Dear Shuchu,
> > what do you think is the problem :the bg is low but you don't have a
> > specific signal or the bg is so high that the specific signal can't
> > be seen?
> > bruno
> >
> > Bruno SAUBAMEA
> >
> > EA 3621 & Service Commun d'Imagerie Cellulaire et Moléculaire
> >
> > Faculté des Sciences Pharmaceutiques et Biologiques
> > Université Paris Descartes
> > 4, avenue de l'Observatoire
> > 75006 PARIS
> >
> > tel : [phone]
> > fax : [phone]
> >   ----- Original Message -----
> >   From: Shuchi Gupta
> >   To: [hidden email]
> >   Sent: Monday, March 09, 2009 5:23 PM
> >   Subject: secondary antibody from immunostainigs
> >
> >
> >   hello,
> >     i want to know if somebody knows any good secondary antibodies(
> >   alexa, atto conjugated) against rabbit which i can use for the
> >   immunostainings. i have tried  antibodies from cell signaling,  
> abnova
> >   and dako.but none of them give me the specific signal .( this i  
> get to
> >   know from my controls which contain only my secondary  
> antibody) .  i
> >   am using these antibodies in mouse platelets.i block them with  
> BSA 2%
> >   after fixing and permeabilization.
> >   hope to get the suggestions soon.
> >   thank you
> >   shuchi
> >

Glen MacDonald
Core for Communication Research
Virginia Merrill Bloedel Hearing Research Center
Box 357923
University of Washington
Seattle, WA 98195-7923  USA
[phone]
[hidden email]

******************************************************************************
The box said "Requires WindowsXP or better", so I bought a Macintosh.
******************************************************************************

[vb-2]

Dear List,

I am looking for feedback on CFM-1 Plus mounting media (with the antifadent;
the latter brings pH to ca. 10), in particular on stability of CFP, CeFP,
YFP, venusYFP, etc.  fluorescence at pH 10.

Thank you,


Vitaly
NCI-Frederick,
301-846-6575
 

[Kilgore, Jason]

 

**VENDOR RESPONSE**

 

Dear Julia,

 

Quite likely the non-specific nuclear background you are seeing is due to charge interactions.  The negatively-charged Alexa Fluor dyes are drawn to the positively-charged nuclei, and sometimes mitochondria.  On brain cryosections this shows up as myelin labeling.  Standard protein blocking won’t combat this.

 

I work at Molecular Probes and, several years ago while I was still running a lab here I helped develop a product called the Image-iT FX Signal Enhancer solution.  This is a blocking reagent for those charge interactions, which comes in a dropper bottle formulation.  Just drop on a couple drops prior to doing the protein blocking step. 

 

http://products.invitrogen.com/ivgn/en/US/adirect/invitrogen?cmd=catProductDetail&productID=I36933

 

Cheers,

 

Jason

 

** Commercial affiliation acknowledged **

 

Jason A. Kilgore

Technical Support Scientist

Molecular Probes Labeling and Detection Technologies

Cells Systems Division

 

29851 Willow Creek Rd.

Eugene, OR  97402-9132

T: 541.335.0353

F: 541.335.0238

 

[hidden email]

www.invitrogen.com

invitrogen, part of life technologies

 

 P Save trees.  Please don't print this e-mail unless you really need to.

 

 

---

From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of js1719
Sent: Monday, March 09, 2009 11:14 AM
To: [hidden email]
Subject: Re: secondary antibody from immunostainigs

 

Are you getting nonspecific background in the nucleus? We often find that the Molecular Probes Alexa in 568 and 594 have significant background labeling of the nuclear region- not sure why. It can be reduced by storing your secondary in aliquoits in the -80C to avoid multiple freeze thaws and to spin the tube at 13K for 10 minutes prior to use. 

good luck

Julia

 

 

On Mar 9, 2009, at 1:34 PM, MODEL, MICHAEL wrote:

It seems to me the problem most likely lies with either primary antibody or sample preparation

 

From: Confocal Microscopy List [[hidden email]] On Behalf Of Dries Vercauteren
Sent: Monday, March 09, 2009 12:45 PM
To: [hidden email]
Subject: Re: secondary antibody from immunostainigs

 

Dear Shuchi,

did you try goat anti rabbit-Alexa Fluor 594 (Mol prob; A11012) yet?

No commercial interest.

Good luck,

Dries

2009/3/9 Shuchi Gupta <[hidden email]>

hello,
 i want to know if somebody knows any good secondary antibodies( alexa, atto conjugated) against rabbit which i can use for the immunostainings. i have tried  antibodies from cell signaling, abnova and dako.but none of them give me the specific signal .( this i get to know from my controls which contain only my secondary antibody) .  i am using these antibodies in mouse platelets.i block them with BSA 2% after fixing and permeabilization.
hope to get the suggestions soon.
thank you
shuchi

-- 
Dries Vercauteren, PhD student
Master of Bioscience Engineering: Cell and Gene Biotechnology

Ghent Research Group on Nanomedicines
www.ugent.be/fw/en/research/biofys
Faculty of pharmaceutical sciences, Ghent University
Harelbekestraat 72, 9000 Ghent
Belgium

Phone:  [phone]
Mobile: [phone]
E-mail: [hidden email]
          [hidden email]

 

[Mario-2]

This is both a test regarding the posted message plus duplicate "we already got this one" which happens to me every time but I have ignored it, ...and a comment regarding platelet labeling.

Shuchi, I may have missed it, but platelet imaging is difficult given their very small size. If mice platelets are anything like human, than typical sizes are ~2-3 um in diameter without any nucleus. Labeling any internal organelles is going to be difficult. Often, 2% BSA blocking is not adequate. Further, I don't recall you mentioning which fixative you used but if it contained glutaraldehyde, you may end up non-specifically crosslinking some of your secondary or turning your platelets fluorescent from Glut. polymers. during fixation. Have you checked your fixed platelets for native fluorescence?

Paraformaldehyde/formaldehyde which is much more membrane permeant than Glut., usually does a better job as a fixative with respects to avoiding aldehyde related fluorescence background. If you are using a cold solvent such as EtOH or acetone as a fixative, it avoids the free aldehyde problem but organelle and plasma membranes might get scrambled including your target.

A blocking agent besides BSA that you might try, and that has worked significantly better for me, is Pierce's SuperBlock in PBS or Tris buffer. I use it for diluting my stock antibodies as well as for washing between antibody applications.

Let us know how things work out; imaging platelets is a challenge for standard confocal microscopy.

Mario

 

---

From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of js1719
Sent: Monday, March 09, 2009 11:14 AM
To: [hidden email]
Subject: Re: secondary antibody from immunostainigs

 

Are you getting nonspecific background in the nucleus? We often find that the Molecular Probes Alexa in 568 and 594 have significant background labeling of the nuclear region- not sure why. It can be reduced by storing your secondary in aliquoits in the -80C to avoid multiple freeze thaws and to spin the tube at 13K for 10 minutes prior to use. 

good luck

Julia

 

 

On Mar 9, 2009, at 1:34 PM, MODEL, MICHAEL wrote:

It seems to me the problem most likely lies with either primary antibody or sample preparation

 

From: Confocal Microscopy List [[hidden email]] On Behalf Of Dries Vercauteren
Sent: Monday, March 09, 2009 12:45 PM
To: [hidden email]
Subject: Re: secondary antibody from immunostainigs

 

Dear Shuchi,

did you try goat anti rabbit-Alexa Fluor 594 (Mol prob; A11012) yet?

No commercial interest.

Good luck,

Dries

2009/3/9 Shuchi Gupta <[hidden email]>

hello,
 i want to know if somebody knows any good secondary antibodies( alexa, atto conjugated) against rabbit which i can use for the immunostainings. i have tried  antibodies from cell signaling, abnova and dako.but none of them give me the specific signal .( this i get to know from my controls which contain only my secondary antibody) .  i am using these antibodies in mouse platelets.i block them with BSA 2% after fixing and permeabilization.
hope to get the suggestions soon.
thank you
shuchi

-- 
Dries Vercauteren, PhD student
Master of Bioscience Engineering: Cell and Gene Biotechnology

Ghent Research Group on Nanomedicines
www.ugent.be/fw/en/research/biofys
Faculty of pharmaceutical sciences, Ghent University
Harelbekestraat 72, 9000 Ghent
Belgium

Phone:  [phone]
Mobile: [phone]
E-mail: [hidden email]
          [hidden email]

 

-- 

________________________________________________________________________________

Mario M. Moronne, Ph.D.

[hidden email]
[hidden email]

[Shuchi Gupta]

Dear Mario ,
thank you so much for your reply. i am using PFA for fixation of my  
spreaded mouse platelets and then block them overnite with 2% BSA and  
rabbit serum (1%)in PBS. i am using rabbit atto 647 as the secondary  
antibody to study the localisation of some proteins. yaa u r right  
mouse platelets are 2-5 um in diameter. and really hard to visualise  
with confocal . but i still want to try as it will be helpful in my  
thesis. i would like to know about the pierce blocking solution you  
mentioned if it  is even good for immunofloresence as the product home  
page doesnt give any details about its use in IF.
waiting to hear from you
thank you
Shuchi Gupta


Quoting Mario <[hidden email]>:

> This is both a test regarding the posted message plus duplicate "we  
> already got this one" which happens to me every time but I have  
> ignored it, ...and a comment regarding platelet labeling.
>
> Shuchi, I may have missed it, but platelet imaging is difficult  
> given their very small size. If mice platelets are anything like  
> human, than typical sizes are ~2-3 um in diameter without any  
> nucleus. Labeling any internal organelles is going to be difficult.  
> Often, 2% BSA blocking is not adequate. Further, I don't recall you  
> mentioning which fixative you used but if it contained  
> glutaraldehyde, you may end up non-specifically crosslinking some of  
> your secondary or turning your platelets fluorescent from Glut.  
> polymers. during fixation. Have you checked your fixed platelets for  
> native fluorescence?
>
> Paraformaldehyde/formaldehyde which is much more membrane permeant  
> than Glut., usually does a better job as a fixative with respects to  
> avoiding aldehyde related fluorescence background. If you are using  
> a cold solvent such as EtOH or acetone as a fixative, it avoids the  
> free aldehyde problem but organelle and plasma membranes might get  
> scrambled including your target.
>
> A blocking agent besides BSA that you might try, and that has worked  
> significantly better for me, is Pierce's SuperBlock in PBS or Tris  
> buffer. I use it for diluting my stock antibodies as well as for  
> washing between antibody applications.
>
> Let us know how things work out; imaging platelets is a challenge  
> for standard confocal microscopy.
>
> Mario
>>
>>
>> From: Confocal Microscopy List  
>> [mailto:[hidden email]] On Behalf Of js1719
>> Sent: Monday, March 09, 2009 11:14 AM
>> To: [hidden email]
>> Subject: Re: secondary antibody from immunostainigs
>>
>> Are you getting nonspecific background in the nucleus? We often  
>> find that the Molecular Probes Alexa in 568 and 594 have  
>> significant background labeling of the nuclear region- not sure  
>> why. It can be reduced by storing your secondary in aliquoits in  
>> the -80C to avoid multiple freeze thaws and to spin the tube at 13K  
>> for 10 minutes prior to use. good luck
>> Julia
>>
>>
>> On Mar 9, 2009, at 1:34 PM, MODEL, MICHAEL wrote:
>>
>>
>> It seems to me the problem most likely lies with either primary  
>> antibody or sample preparation
>>
>> From: Confocal Microscopy List  
>> [<mailto:[hidden email]>mailto:[hidden email]] On Behalf Of Dries  
>> Vercauteren
>> Sent: Monday, March 09, 2009 12:45 PM
>> To:  
>> <mailto:[hidden email]>[hidden email]
>> Subject: Re: secondary antibody from immunostainigs
>>
>> Dear Shuchi,
>>
>> did you try goat anti rabbit-Alexa Fluor 594 (Mol prob; A11012) yet?
>>
>> No commercial interest.
>>
>> Good luck,
>>
>> Dries
>> 2009/3/9 Shuchi Gupta  
>> <<mailto:[hidden email]>[hidden email]>
>> hello,
>> i want to know if somebody knows any good secondary antibodies(  
>> alexa, atto conjugated) against rabbit which i can use for the  
>> immunostainings. i have tried  antibodies from cell signaling,  
>> abnova and dako.but none of them give me the specific signal .(  
>> this i get to know from my controls which contain only my secondary  
>> antibody) .  i am using these antibodies in mouse platelets.i block  
>> them with BSA 2% after fixing and permeabilization.
>> hope to get the suggestions soon.
>> thank you
>> shuchi
>>
>>
>>
>> --
>> Dries Vercauteren, PhD student
>> Master of Bioscience Engineering: Cell and Gene Biotechnology
>>
>> Ghent Research Group on Nanomedicines
>> <http://www.ugent.be/fw/en/research/biofys>www.ugent.be/fw/en/research/biofys
>> Faculty of pharmaceutical sciences, Ghent University
>> Harelbekestraat 72, 9000 Ghent
>> Belgium
>>
>> Phone:  [phone]
>> Mobile: [phone]
>> E-mail: <mailto:[hidden email]>[hidden email]
>>          <mailto:[hidden email]>[hidden email]
>>
>
>
> --
> ________________________________________________________________________________
> Mario M. Moronne, Ph.D.
>
> [hidden email]
> [hidden email]
>

[jlribas]

nº de bastidor: W0L0AHM758G160994

He hablado con ellos y no van a llevaros el coche. Dicen que normalmente
van los peritos al concesionario.


Shuchi Gupta escribió:

> Dear Mario ,
> thank you so much for your reply. i am using PFA for fixation of my
> spreaded mouse platelets and then block them overnite with 2% BSA and
> rabbit serum (1%)in PBS. i am using rabbit atto 647 as the secondary
> antibody to study the localisation of some proteins. yaa u r right
> mouse platelets are 2-5 um in diameter. and really hard to visualise
> with confocal . but i still want to try as it will be helpful in my
> thesis. i would like to know about the pierce blocking solution you
> mentioned if it  is even good for immunofloresence as the product home
> page doesnt give any details about its use in IF.
> waiting to hear from you
> thank you
> Shuchi Gupta
>
>
> Quoting Mario <[hidden email]>:
>
>> This is both a test regarding the posted message plus duplicate "we
>> already got this one" which happens to me every time but I have
>> ignored it, ...and a comment regarding platelet labeling.
>>
>> Shuchi, I may have missed it, but platelet imaging is difficult given
>> their very small size. If mice platelets are anything like human,
>> than typical sizes are ~2-3 um in diameter without any nucleus.
>> Labeling any internal organelles is going to be difficult. Often, 2%
>> BSA blocking is not adequate. Further, I don't recall you mentioning
>> which fixative you used but if it contained glutaraldehyde, you may
>> end up non-specifically crosslinking some of your secondary or
>> turning your platelets fluorescent from Glut. polymers. during
>> fixation. Have you checked your fixed platelets for native fluorescence?
>>
>> Paraformaldehyde/formaldehyde which is much more membrane permeant
>> than Glut., usually does a better job as a fixative with respects to
>> avoiding aldehyde related fluorescence background. If you are using a
>> cold solvent such as EtOH or acetone as a fixative, it avoids the
>> free aldehyde problem but organelle and plasma membranes might get
>> scrambled including your target.
>>
>> A blocking agent besides BSA that you might try, and that has worked
>> significantly better for me, is Pierce's SuperBlock in PBS or Tris
>> buffer. I use it for diluting my stock antibodies as well as for
>> washing between antibody applications.
>>
>> Let us know how things work out; imaging platelets is a challenge for
>> standard confocal microscopy.
>>
>> Mario
>>>
>>>
>>> From: Confocal Microscopy List
>>> [mailto:[hidden email]] On Behalf Of js1719
>>> Sent: Monday, March 09, 2009 11:14 AM
>>> To: [hidden email]
>>> Subject: Re: secondary antibody from immunostainigs
>>>
>>> Are you getting nonspecific background in the nucleus? We often find
>>> that the Molecular Probes Alexa in 568 and 594 have significant
>>> background labeling of the nuclear region- not sure why. It can be
>>> reduced by storing your secondary in aliquoits in the -80C to avoid
>>> multiple freeze thaws and to spin the tube at 13K for 10 minutes
>>> prior to use. good luck
>>> Julia
>>>
>>>
>>> On Mar 9, 2009, at 1:34 PM, MODEL, MICHAEL wrote:
>>>
>>>
>>> It seems to me the problem most likely lies with either primary
>>> antibody or sample preparation
>>>
>>> From: Confocal Microscopy List
>>> [<mailto:[hidden email]>mailto:[hidden email]]
>>> On Behalf Of Dries Vercauteren
>>> Sent: Monday, March 09, 2009 12:45 PM
>>> To:
>>> <mailto:[hidden email]>[hidden email]
>>>
>>> Subject: Re: secondary antibody from immunostainigs
>>>
>>> Dear Shuchi,
>>>
>>> did you try goat anti rabbit-Alexa Fluor 594 (Mol prob; A11012) yet?
>>>
>>> No commercial interest.
>>>
>>> Good luck,
>>>
>>> Dries
>>> 2009/3/9 Shuchi Gupta
>>> <<mailto:[hidden email]>[hidden email]>
>>>
>>> hello,
>>> i want to know if somebody knows any good secondary antibodies(
>>> alexa, atto conjugated) against rabbit which i can use for the
>>> immunostainings. i have tried  antibodies from cell signaling,
>>> abnova and dako.but none of them give me the specific signal .( this
>>> i get to know from my controls which contain only my secondary
>>> antibody) .  i am using these antibodies in mouse platelets.i block
>>> them with BSA 2% after fixing and permeabilization.
>>> hope to get the suggestions soon.
>>> thank you
>>> shuchi
>>>
>>>
>>>
>>> --
>>> Dries Vercauteren, PhD student
>>> Master of Bioscience Engineering: Cell and Gene Biotechnology
>>>
>>> Ghent Research Group on Nanomedicines
>>> <http://www.ugent.be/fw/en/research/biofys>www.ugent.be/fw/en/research/biofys
>>>
>>> Faculty of pharmaceutical sciences, Ghent University
>>> Harelbekestraat 72, 9000 Ghent
>>> Belgium
>>>
>>> Phone:  [phone]
>>> Mobile: [phone]
>>> E-mail: <mailto:[hidden email]>[hidden email]
>>>          <mailto:[hidden email]>[hidden email]
>>>
>>
>>
>> --
>> ________________________________________________________________________________
>>
>> Mario M. Moronne, Ph.D.
>>
>> [hidden email]
>> [hidden email]
>>
>

--
Juan Luis Ribas
Servicio de Microscopía
Centro de Investigación, Tecnología e Innovación
Universidad de Sevilla
Av. Reina Mercedes 4b
41012 Sevilla

Tfno: 954559983

[jlribas]

To the administrator of the list:

This e-mail has been a mistake from me. Please don't distribute it to
the list.

Sorry for the mistake.

Best regards

Juan Luis Ribas escribió:

> nº de bastidor: W0L0AHM758G160994
>
> He hablado con ellos y no van a llevaros el coche. Dicen que
> normalmente van los peritos al concesionario.
>
>
> Shuchi Gupta escribió:
>> Dear Mario ,
>> thank you so much for your reply. i am using PFA for fixation of my
>> spreaded mouse platelets and then block them overnite with 2% BSA and
>> rabbit serum (1%)in PBS. i am using rabbit atto 647 as the secondary
>> antibody to study the localisation of some proteins. yaa u r right
>> mouse platelets are 2-5 um in diameter. and really hard to visualise
>> with confocal . but i still want to try as it will be helpful in my
>> thesis. i would like to know about the pierce blocking solution you
>> mentioned if it  is even good for immunofloresence as the product
>> home page doesnt give any details about its use in IF.
>> waiting to hear from you
>> thank you
>> Shuchi Gupta
>>
>>
>> Quoting Mario <[hidden email]>:
>>
>>> This is both a test regarding the posted message plus duplicate "we
>>> already got this one" which happens to me every time but I have
>>> ignored it, ...and a comment regarding platelet labeling.
>>>
>>> Shuchi, I may have missed it, but platelet imaging is difficult
>>> given their very small size. If mice platelets are anything like
>>> human, than typical sizes are ~2-3 um in diameter without any
>>> nucleus. Labeling any internal organelles is going to be difficult.
>>> Often, 2% BSA blocking is not adequate. Further, I don't recall you
>>> mentioning which fixative you used but if it contained
>>> glutaraldehyde, you may end up non-specifically crosslinking some of
>>> your secondary or turning your platelets fluorescent from Glut.
>>> polymers. during fixation. Have you checked your fixed platelets for
>>> native fluorescence?
>>>
>>> Paraformaldehyde/formaldehyde which is much more membrane permeant
>>> than Glut., usually does a better job as a fixative with respects to
>>> avoiding aldehyde related fluorescence background. If you are using
>>> a cold solvent such as EtOH or acetone as a fixative, it avoids the
>>> free aldehyde problem but organelle and plasma membranes might get
>>> scrambled including your target.
>>>
>>> A blocking agent besides BSA that you might try, and that has worked
>>> significantly better for me, is Pierce's SuperBlock in PBS or Tris
>>> buffer. I use it for diluting my stock antibodies as well as for
>>> washing between antibody applications.
>>>
>>> Let us know how things work out; imaging platelets is a challenge
>>> for standard confocal microscopy.
>>>
>>> Mario
>>>>
>>>>
>>>> From: Confocal Microscopy List
>>>> [mailto:[hidden email]] On Behalf Of js1719
>>>> Sent: Monday, March 09, 2009 11:14 AM
>>>> To: [hidden email]
>>>> Subject: Re: secondary antibody from immunostainigs
>>>>
>>>> Are you getting nonspecific background in the nucleus? We often
>>>> find that the Molecular Probes Alexa in 568 and 594 have
>>>> significant background labeling of the nuclear region- not sure
>>>> why. It can be reduced by storing your secondary in aliquoits in
>>>> the -80C to avoid multiple freeze thaws and to spin the tube at 13K
>>>> for 10 minutes prior to use. good luck
>>>> Julia
>>>>
>>>>
>>>> On Mar 9, 2009, at 1:34 PM, MODEL, MICHAEL wrote:
>>>>
>>>>
>>>> It seems to me the problem most likely lies with either primary
>>>> antibody or sample preparation
>>>>
>>>> From: Confocal Microscopy List
>>>> [<mailto:[hidden email]>mailto:[hidden email]]
>>>> On Behalf Of Dries Vercauteren
>>>> Sent: Monday, March 09, 2009 12:45 PM
>>>> To:
>>>> <mailto:[hidden email]>[hidden email]
>>>>
>>>> Subject: Re: secondary antibody from immunostainigs
>>>>
>>>> Dear Shuchi,
>>>>
>>>> did you try goat anti rabbit-Alexa Fluor 594 (Mol prob; A11012) yet?
>>>>
>>>> No commercial interest.
>>>>
>>>> Good luck,
>>>>
>>>> Dries
>>>> 2009/3/9 Shuchi Gupta
>>>> <<mailto:[hidden email]>[hidden email]>
>>>>
>>>> hello,
>>>> i want to know if somebody knows any good secondary antibodies(
>>>> alexa, atto conjugated) against rabbit which i can use for the
>>>> immunostainings. i have tried  antibodies from cell signaling,
>>>> abnova and dako.but none of them give me the specific signal .(
>>>> this i get to know from my controls which contain only my secondary
>>>> antibody) .  i am using these antibodies in mouse platelets.i block
>>>> them with BSA 2% after fixing and permeabilization.
>>>> hope to get the suggestions soon.
>>>> thank you
>>>> shuchi
>>>>
>>>>
>>>>
>>>> --
>>>> Dries Vercauteren, PhD student
>>>> Master of Bioscience Engineering: Cell and Gene Biotechnology
>>>>
>>>> Ghent Research Group on Nanomedicines
>>>> <http://www.ugent.be/fw/en/research/biofys>www.ugent.be/fw/en/research/biofys
>>>>
>>>> Faculty of pharmaceutical sciences, Ghent University
>>>> Harelbekestraat 72, 9000 Ghent
>>>> Belgium
>>>>
>>>> Phone:  [phone]
>>>> Mobile: [phone]
>>>> E-mail: <mailto:[hidden email]>[hidden email]
>>>>          <mailto:[hidden email]>[hidden email]
>>>>
>>>
>>>
>>> --
>>> ________________________________________________________________________________
>>>
>>> Mario M. Moronne, Ph.D.
>>>
>>> [hidden email]
>>> [hidden email]
>>>
>>
>

--
Juan Luis Ribas
Servicio de Microscopía
Centro de Investigación, Tecnología e Innovación
Universidad de Sevilla
Av. Reina Mercedes 4b
41012 Sevilla

Tfno: 954559983

[anh2006]

What is "rabbit atto 647"? Your protocol still isn't clear to me so it is hard to help.  You should be able to get rid of background easily - we stain megas and platelets with no background with no special tricks. Adding and subtracting blocking buffers will only work once you isolate your problem, which I am pretty sure hasn't been done yet.

Your platelets are murine, correct? What species if you primary and what sppecies is your secondary? As I asked before, are you staining tissues or in vitro platelet preps? If tissue, is your secondary cross adsorbed so it won't bind to mouse IgG?



-----Original Message-----
From: Shuchi Gupta <[hidden email]>

Date: Thu, 12 Mar 2009 10:15:43
To: <[hidden email]>
Subject: Re: Secondary antibody comment plus test regarding spurious posting
 rejection


Dear Mario ,
thank you so much for your reply. i am using PFA for fixation of my  
spreaded mouse platelets and then block them overnite with 2% BSA and  
rabbit serum (1%)in PBS. i am using rabbit atto 647 as the secondary  
antibody to study the localisation of some proteins. yaa u r right  
mouse platelets are 2-5 um in diameter. and really hard to visualise  
with confocal . but i still want to try as it will be helpful in my  
thesis. i would like to know about the pierce blocking solution you  
mentioned if it  is even good for immunofloresence as the product home  
page doesnt give any details about its use in IF.
waiting to hear from you
thank you
Shuchi Gupta


Quoting Mario <[hidden email]>:

> This is both a test regarding the posted message plus duplicate "we  
> already got this one" which happens to me every time but I have  
> ignored it, ...and a comment regarding platelet labeling.
>
> Shuchi, I may have missed it, but platelet imaging is difficult  
> given their very small size. If mice platelets are anything like  
> human, than typical sizes are ~2-3 um in diameter without any  
> nucleus. Labeling any internal organelles is going to be difficult.  
> Often, 2% BSA blocking is not adequate. Further, I don't recall you  
> mentioning which fixative you used but if it contained  
> glutaraldehyde, you may end up non-specifically crosslinking some of  
> your secondary or turning your platelets fluorescent from Glut.  
> polymers. during fixation. Have you checked your fixed platelets for  
> native fluorescence?
>
> Paraformaldehyde/formaldehyde which is much more membrane permeant  
> than Glut., usually does a better job as a fixative with respects to  
> avoiding aldehyde related fluorescence background. If you are using  
> a cold solvent such as EtOH or acetone as a fixative, it avoids the  
> free aldehyde problem but organelle and plasma membranes might get  
> scrambled including your target.
>
> A blocking agent besides BSA that you might try, and that has worked  
> significantly better for me, is Pierce's SuperBlock in PBS or Tris  
> buffer. I use it for diluting my stock antibodies as well as for  
> washing between antibody applications.
>
> Let us know how things work out; imaging platelets is a challenge  
> for standard confocal microscopy.
>
> Mario
>>
>>
>> From: Confocal Microscopy List  
>> [mailto:[hidden email]] On Behalf Of js1719
>> Sent: Monday, March 09, 2009 11:14 AM
>> To: [hidden email]
>> Subject: Re: secondary antibody from immunostainigs
>>
>> Are you getting nonspecific background in the nucleus? We often  
>> find that the Molecular Probes Alexa in 568 and 594 have  
>> significant background labeling of the nuclear region- not sure  
>> why. It can be reduced by storing your secondary in aliquoits in  
>> the -80C to avoid multiple freeze thaws and to spin the tube at 13K  
>> for 10 minutes prior to use. good luck
>> Julia
>>
>>
>> On Mar 9, 2009, at 1:34 PM, MODEL, MICHAEL wrote:
>>
>>
>> It seems to me the problem most likely lies with either primary  
>> antibody or sample preparation
>>
>> From: Confocal Microscopy List  
>> [<mailto:[hidden email]>mailto:[hidden email]] On Behalf Of Dries  
>> Vercauteren
>> Sent: Monday, March 09, 2009 12:45 PM
>> To:  
>> <mailto:[hidden email]>[hidden email]
>> Subject: Re: secondary antibody from immunostainigs
>>
>> Dear Shuchi,
>>
>> did you try goat anti rabbit-Alexa Fluor 594 (Mol prob; A11012) yet?
>>
>> No commercial interest.
>>
>> Good luck,
>>
>> Dries
>> 2009/3/9 Shuchi Gupta  
>> <<mailto:[hidden email]>[hidden email]>
>> hello,
>> i want to know if somebody knows any good secondary antibodies(  
>> alexa, atto conjugated) against rabbit which i can use for the  
>> immunostainings. i have tried  antibodies from cell signaling,  
>> abnova and dako.but none of them give me the specific signal .(  
>> this i get to know from my controls which contain only my secondary  
>> antibody) .  i am using these antibodies in mouse platelets.i block  
>> them with BSA 2% after fixing and permeabilization.
>> hope to get the suggestions soon.
>> thank you
>> shuchi
>>
>>
>>
>> --
>> Dries Vercauteren, PhD student
>> Master of Bioscience Engineering: Cell and Gene Biotechnology
>>
>> Ghent Research Group on Nanomedicines
>> <http://www.ugent.be/fw/en/research/biofys>www.ugent.be/fw/en/research/biofys
>> Faculty of pharmaceutical sciences, Ghent University
>> Harelbekestraat 72, 9000 Ghent
>> Belgium
>>
>> Phone:  [phone]
>> Mobile: [phone]
>> E-mail: <mailto:[hidden email]>[hidden email]
>>          <mailto:[hidden email]>[hidden email]
>>
>
>
> --
>________________________________________________________________________________
> Mario M. Moronne, Ph.D.
>
> [hidden email]
> [hidden email]
>

[Bruno Saubaméa]

Dear Shushi

I might have not understood correctly but it seems that your primary antibody is made in rabbit (since you use anti rabbit secondary Ab). So the problem of non specific staining probably comes from the rabbit serum that you use to block. You should block with a serum from the same species that your secondary Ab is made from (goat or donkey probably)

bruno

 

Bruno SAUBAMEA

 

EA 3621 & Service Commun d'Imagerie Cellulaire et Moléculaire

 

Faculté des Sciences Pharmaceutiques et Biologiques
Université Paris Descartes
4, avenue de l'Observatoire
75006 PARIS

 

tel : [phone]
fax : [phone]

----- Original Message -----

From: [hidden email]

To: [hidden email]

Sent: Thursday, March 12, 2009 10:15 AM

Subject: Re: Secondary antibody comment plus test regarding spurious posting rejection

Dear Mario ,
thank you so much for your reply. i am using PFA for fixation of my 
spreaded mouse platelets and then block them overnite with 2% BSA and 
rabbit serum (1%)in PBS. i am using rabbit atto 647 as the secondary 
antibody to study the localisation of some proteins. yaa u r right 
mouse platelets are 2-5 um in diameter. and really hard to visualise 
with confocal . but i still want to try as it will be helpful in my 
thesis. i would like to know about the pierce blocking solution you 
mentioned if it  is even good for immunofloresence as the product home 
page doesnt give any details about its use in IF.
waiting to hear from you
thank you
Shuchi Gupta


Quoting Mario <[hidden email]>:

> This is both a test regarding the posted message plus duplicate "we 
> already got this one" which happens to me every time but I have 
> ignored it, ...and a comment regarding platelet labeling.
>
> Shuchi, I may have missed it, but platelet imaging is difficult 
> given their very small size. If mice platelets are anything like 
> human, than typical sizes are ~2-3 um in diameter without any 
> nucleus. Labeling any internal organelles is going to be difficult. 
> Often, 2% BSA blocking is not adequate. Further, I don't recall you 
> mentioning which fixative you used but if it contained 
> glutaraldehyde, you may end up non-specifically crosslinking some of 
> your secondary or turning your platelets fluorescent from Glut. 
> polymers. during fixation. Have you checked your fixed platelets for 
> native fluorescence?
>
> Paraformaldehyde/formaldehyde which is much more membrane permeant 
> than Glut., usually does a better job as a fixative with respects to 
> avoiding aldehyde related fluorescence background. If you are using 
> a cold solvent such as EtOH or acetone as a fixative, it avoids the 
> free aldehyde problem but organelle and plasma membranes might get 
> scrambled including your target.
>
> A blocking agent besides BSA that you might try, and that has worked 
> significantly better for me, is Pierce's SuperBlock in PBS or Tris 
> buffer. I use it for diluting my stock antibodies as well as for 
> washing between antibody applications.
>
> Let us know how things work out; imaging platelets is a challenge 
> for standard confocal microscopy.
>
> Mario
>>
>>
>> From: Confocal Microscopy List 
>> [mailto:[hidden email]] On Behalf Of js1719
>> Sent: Monday, March 09, 2009 11:14 AM
>> To: [hidden email]
>> Subject: Re: secondary antibody from immunostainigs
>>
>> Are you getting nonspecific background in the nucleus? We often 
>> find that the Molecular Probes Alexa in 568 and 594 have 
>> significant background labeling of the nuclear region- not sure 
>> why. It can be reduced by storing your secondary in aliquoits in 
>> the -80C to avoid multiple freeze thaws and to spin the tube at 13K 
>> for 10 minutes prior to use. good luck
>> Julia
>>
>>
>> On Mar 9, 2009, at 1:34 PM, MODEL, MICHAEL wrote:
>>
>>
>> It seems to me the problem most likely lies with either primary 
>> antibody or sample preparation
>>
>> From: Confocal Microscopy List 
>> [<[hidden email]] On Behalf Of Dries 
>> Vercauteren
>> Sent: Monday, March 09, 2009 12:45 PM
>> To: 
>> <[hidden email]
>> Subject: Re: secondary antibody from immunostainigs
>>
>> Dear Shuchi,
>>
>> did you try goat anti rabbit-Alexa Fluor 594 (Mol prob; A11012) yet?
>>
>> No commercial interest.
>>
>> Good luck,
>>
>> Dries
>> 2009/3/9 Shuchi Gupta 
>> <<[hidden email]>
>> hello,
>> i want to know if somebody knows any good secondary antibodies( 
>> alexa, atto conjugated) against rabbit which i can use for the 
>> immunostainings. i have tried  antibodies from cell signaling, 
>> abnova and dako.but none of them give me the specific signal .( 
>> this i get to know from my controls which contain only my secondary 
>> antibody) .  i am using these antibodies in mouse platelets.i block 
>> them with BSA 2% after fixing and permeabilization.
>> hope to get the suggestions soon.
>> thank you
>> shuchi
>>
>>
>>
>> --
>> Dries Vercauteren, PhD student
>> Master of Bioscience Engineering: Cell and Gene Biotechnology
>>
>> Ghent Research Group on Nanomedicines
>> <<A href="http://www.ugent.be/fw/en/research/biofys>www.ugent.be/fw/en/research/biofys">http://www.ugent.be/fw/en/research/biofys>www.ugent.be/fw/en/research/biofys
>> Faculty of pharmaceutical sciences, Ghent University
>> Harelbekestraat 72, 9000 Ghent
>> Belgium
>>
>> Phone:  [phone]
>> Mobile: [phone]
>> E-mail: <[hidden email]
>>          <[hidden email]
>>
>
>
> --
> ________________________________________________________________________________
> Mario M. Moronne, Ph.D.
>
> [hidden email]
> [hidden email]
>

[anh2006]

I see .... If your primary is raised in rabbit and you are using an anti-rabbit secondary you should not be using rabbit IgG to block. That is the fatal flaw of your protocol. Either eliminate the serum or use the serum from the host of your secondary (in your case goat IgG).

-----Original Message-----
From: Shuchi Gupta <[hidden email]>

Date: Thu, 12 Mar 2009 16:02:26
To: <[hidden email]>
Subject: Re: Secondary antibody comment plus test regarding spurious
        postingrejection





hi
i am staining the mouse platelets which i spread after isolation from  
the blood. i fix them in PFA and block them overnite at in cold which  
2% BSA. my primary antibodies are raised in rabbit and thus using  
secondary antibodies which are anti rabbit ( atto 647) and raised in  
goat. but i am not able to see any specific staining for my proteins.  
it is either no signal or it is all background with very less signal.  
so i am not able to judge that it is no binding of my primany antibody  
to the protein of interest or it is some other problem. i hope i am  
making things clear now.
thank you
Shuchi Gupta

Quoting [hidden email]:

> What is "rabbit atto 647"? Your protocol still isn't clear to me so  
> it is hard to help.  You should be able to get rid of background  
> easily - we stain megas and platelets with no background with no  
> special tricks. Adding and subtracting blocking buffers will only  
> work once you isolate your problem, which I am pretty sure hasn't  
> been done yet.
>
> Your platelets are murine, correct? What species if you primary and  
> what sppecies is your secondary? As I asked before, are you staining  
> tissues or in vitro platelet preps? If tissue, is your secondary  
> cross adsorbed so it won't bind to mouse IgG?
>
>
>
> -----Original Message-----
> From: Shuchi Gupta <[hidden email]>
>
> Date: Thu, 12 Mar 2009 10:15:43
> To: <[hidden email]>
> Subject: Re: Secondary antibody comment plus test regarding spurious posting
>  rejection
>
>
> Dear Mario ,
> thank you so much for your reply. i am using PFA for fixation of my
> spreaded mouse platelets and then block them overnite with 2% BSA and
> rabbit serum (1%)in PBS. i am using rabbit atto 647 as the secondary
> antibody to study the localisation of some proteins. yaa u r right
> mouse platelets are 2-5 um in diameter. and really hard to visualise
> with confocal . but i still want to try as it will be helpful in my
> thesis. i would like to know about the pierce blocking solution you
> mentioned if it  is even good for immunofloresence as the product home
> page doesnt give any details about its use in IF.
> waiting to hear from you
> thank you
> Shuchi Gupta
>
>
> Quoting Mario <[hidden email]>:
>
>> This is both a test regarding the posted message plus duplicate "we
>> already got this one" which happens to me every time but I have
>> ignored it, ...and a comment regarding platelet labeling.
>>
>> Shuchi, I may have missed it, but platelet imaging is difficult
>> given their very small size. If mice platelets are anything like
>> human, than typical sizes are ~2-3 um in diameter without any
>> nucleus. Labeling any internal organelles is going to be difficult.
>> Often, 2% BSA blocking is not adequate. Further, I don't recall you
>> mentioning which fixative you used but if it contained
>> glutaraldehyde, you may end up non-specifically crosslinking some of
>> your secondary or turning your platelets fluorescent from Glut.
>> polymers. during fixation. Have you checked your fixed platelets for
>> native fluorescence?
>>
>> Paraformaldehyde/formaldehyde which is much more membrane permeant
>> than Glut., usually does a better job as a fixative with respects to
>> avoiding aldehyde related fluorescence background. If you are using
>> a cold solvent such as EtOH or acetone as a fixative, it avoids the
>> free aldehyde problem but organelle and plasma membranes might get
>> scrambled including your target.
>>
>> A blocking agent besides BSA that you might try, and that has worked
>> significantly better for me, is Pierce's SuperBlock in PBS or Tris
>> buffer. I use it for diluting my stock antibodies as well as for
>> washing between antibody applications.
>>
>> Let us know how things work out; imaging platelets is a challenge
>> for standard confocal microscopy.
>>
>> Mario
>>>
>>>
>>> From: Confocal Microscopy List
>>> [mailto:[hidden email]] On Behalf Of js1719
>>> Sent: Monday, March 09, 2009 11:14 AM
>>> To: [hidden email]
>>> Subject: Re: secondary antibody from immunostainigs
>>>
>>> Are you getting nonspecific background in the nucleus? We often
>>> find that the Molecular Probes Alexa in 568 and 594 have
>>> significant background labeling of the nuclear region- not sure
>>> why. It can be reduced by storing your secondary in aliquoits in
>>> the -80C to avoid multiple freeze thaws and to spin the tube at 13K
>>> for 10 minutes prior to use. good luck
>>> Julia
>>>
>>>
>>> On Mar 9, 2009, at 1:34 PM, MODEL, MICHAEL wrote:
>>>
>>>
>>> It seems to me the problem most likely lies with either primary
>>> antibody or sample preparation
>>>
>>> From: Confocal Microscopy List
>>> [<mailto:[hidden email]>mailto:[hidden email]] On Behalf Of  
>>> Dries
>>> Vercauteren
>>> Sent: Monday, March 09, 2009 12:45 PM
>>> To:
>>> <mailto:[hidden email]>[hidden email]
>>> Subject: Re: secondary antibody from immunostainigs
>>>
>>> Dear Shuchi,
>>>
>>> did you try goat anti rabbit-Alexa Fluor 594 (Mol prob; A11012) yet?
>>>
>>> No commercial interest.
>>>
>>> Good luck,
>>>
>>> Dries
>>> 2009/3/9 Shuchi Gupta
>>> <<mailto:[hidden email]>[hidden email]>
>>> hello,
>>> i want to know if somebody knows any good secondary antibodies(
>>> alexa, atto conjugated) against rabbit which i can use for the
>>> immunostainings. i have tried  antibodies from cell signaling,
>>> abnova and dako.but none of them give me the specific signal .(
>>> this i get to know from my controls which contain only my secondary
>>> antibody) .  i am using these antibodies in mouse platelets.i block
>>> them with BSA 2% after fixing and permeabilization.
>>> hope to get the suggestions soon.
>>> thank you
>>> shuchi
>>>
>>>
>>>
>>> --
>>> Dries Vercauteren, PhD student
>>> Master of Bioscience Engineering: Cell and Gene Biotechnology
>>>
>>> Ghent Research Group on Nanomedicines
>>> <http://www.ugent.be/fw/en/research/biofys>www.ugent.be/fw/en/research/biofys
>>> Faculty of pharmaceutical sciences, Ghent University
>>> Harelbekestraat 72, 9000 Ghent
>>> Belgium
>>>
>>> Phone:  [phone]
>>> Mobile: [phone]
>>> E-mail: <mailto:[hidden email]>[hidden email]
>>>          <mailto:[hidden email]>[hidden email]
>>>
>>
>>
>> --
>>________________________________________________________________________________
>> Mario M. Moronne, Ph.D.
>>
>> [hidden email]
>> [hidden email]
>>
>

[Ignatius, Mike]

All,

 

No idea why this was reposted - sorry.  Just appeared in my inbox. 

 

Another glitch? 

 

Mike

 

On March 12:

Your message dated Mon, 9 Mar 2009 17:00:38 -0400 with subject "Re:

secondary antibody from immunostainings ***Vendor Response***" has been

successfully distributed to the CONFOCALMICROSCOPY list (2083 recipients).

 

ON March 9th:

Your message dated Mon, 9 Mar 2009 17:00:38 -0400 with subject "Re:

secondary antibody from immunostainings ***Vendor Response***" has been

successfully distributed to the CONFOCALMICROSCOPY list (2080 recipients).

---

From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Ignatius, Mike
Sent: Monday, March 09, 2009 2:01 PM
To: [hidden email]
Subject: Re: secondary antibody from immunostainings ***Vendor Response***

We sell a product, Image-iT FX Signal Enhancer, that was designed to alleviate this type of non-specific binding.  It works especially well with background labeling of the nucleus (and myelin) that can occur with many dyes including the Alexa Fluor line.  The product information sheet or PIS available on the web for this product details what dyes it works best with, and includes the aforementioned 568 and 594.

 

Long name, but short protocol - 30 mins at RT.  Use it in addition to protein blockers and the like, though some papers report seeing it work without additional protein blocking steps - just depends on the source of non-specific signal. 

 

For non-specific, background labeling with Alexa Fluor labeled secondaries, the noise can be reduced by as much as 90%.  For rare event detection and thus long signal integration times it can be very helpful.

 

Mike Ignatius

 

Molecular Probes/Life Technology

---

From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of js1719
Sent: Monday, March 09, 2009 11:14 AM
To: [hidden email]
Subject: Re: secondary antibody from immunostainigs

Are you getting nonspecific background in the nucleus? We often find that the Molecular Probes Alexa in 568 and 594 have significant background labeling of the nuclear region- not sure why. It can be reduced by storing your secondary in aliquoits in the -80C to avoid multiple freeze thaws and to spin the tube at 13K for 10 minutes prior to use. 

good luck

Julia

On Mar 9, 2009, at 1:34 PM, MODEL, MICHAEL wrote:

It seems to me the problem most likely lies with either primary antibody or sample preparation

From: Confocal Microscopy List [[hidden email]] On Behalf Of Dries Vercauteren
Sent: Monday, March 09, 2009 12:45 PM
To: [hidden email]
Subject: Re: secondary antibody from immunostainigs

Dear Shuchi,

did you try goat anti rabbit-Alexa Fluor 594 (Mol prob; A11012) yet?

No commercial interest.

Good luck,

Dries

2009/3/9 Shuchi Gupta <[hidden email]>

hello,
 i want to know if somebody knows any good secondary antibodies( alexa, atto conjugated) against rabbit which i can use for the immunostainings. i have tried  antibodies from cell signaling, abnova and dako.but none of them give me the specific signal .( this i get to know from my controls which contain only my secondary antibody) .  i am using these antibodies in mouse platelets.i block them with BSA 2% after fixing and permeabilization.
hope to get the suggestions soon.
thank you
shuchi

-- 
Dries Vercauteren, PhD student
Master of Bioscience Engineering: Cell and Gene Biotechnology

Ghent Research Group on Nanomedicines
www.ugent.be/fw/en/research/biofys
Faculty of pharmaceutical sciences, Ghent University
Harelbekestraat 72, 9000 Ghent
Belgium

Phone:  [phone]
Mobile: [phone]
E-mail: [hidden email]
          [hidden email]