Blocking in immunocytochemsitry - ImageIT FX Enhancer?

[lechristophe]

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Hi,

Not a confocal question, but a lot of people are doing immuno here so I
thought I'd ask. We are currently using gelatin as the main blocker in our
imunocytochemistry protocol. I was wondering if we could get better
staining (less background / less non specific labeling) by switching to
something else and found Life Technologie's ImageIT FX Enhancer (
http://products.invitrogen.com/ivgn/product/I36933). Does someone have
experience with this, and is the added cost justified? I try to stay clear
of proprietary formulas but sometimes (see Prolong Gold) a "secret" product
does work better.

Thanks for the advices,

Christophe

--
Christophe Leterrier
Researcher
Axonal Domains Architecture Team
CRN2M CNRS UMR 7286
Aix Marseille University, France

[Kilgore, Jason-2]

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** Vendor Reply **

Hi, Christophe,

I actually helped develop that product.  The Image-iT FX Signal Enhancer solution isn't meant to replace protein blocking, and won't work as well as a substitute.  Most dyes have a negative charge, which makes them nonspecifically attracted to positively-charged areas on cells and tissues (in cultured cells, that's mainly the nuclei and mitochondria.  Brain tissue cryosections, it's the myelin).  The Signal Enhancer blocks the positive charges.  But you should still do a subsequent protein blocking.  Not everyone needs it, but if you see background with a secondary-only control that includes a protein blocker, then you might want to add it as a step.  I got to where I used it with every ICC / IHC protocol.

I've never found gelatin to be very effective in my assays as a protein blocker.  I generally recommend 6% Bovine Serum Albumin / 10% normal serum / PBS.  Another commercial option (one of those "proprietary" things you avoid) is called BlockAid, from Life Technologies, which is a mix of various protein blockers.  Use it undiluted or lightly diluted as a blocker and in with your primary.  I've found it to be as good or better than any other common blocking option I've tried for ICC / IHC (even though we market it for blocking microspheres).

Cheers,

Jason

Jason A. Kilgore
Technical Application Scientist
Molecular Probes Labeling and Detection Technologies
Cells Systems Division
 
T 1 800 955 6288 then option 4, then option 6,  or  541 335 0353 * F 541 335 0238
29851 Willow Creek Rd * Eugene * OR * 97402-9132 * United States
www.invitrogen.com/technicalsupport


-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Christophe Leterrier
Sent: Thursday, March 21, 2013 2:24 AM
To: [hidden email]
Subject: Blocking in immunocytochemsitry - ImageIT FX Enhancer?

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
*****

Hi,

Not a confocal question, but a lot of people are doing immuno here so I
thought I'd ask. We are currently using gelatin as the main blocker in our
imunocytochemistry protocol. I was wondering if we could get better
staining (less background / less non specific labeling) by switching to
something else and found Life Technologie's ImageIT FX Enhancer (
http://products.invitrogen.com/ivgn/product/I36933). Does someone have
experience with this, and is the added cost justified? I try to stay clear
of proprietary formulas but sometimes (see Prolong Gold) a "secret" product
does work better.

Thanks for the advices,

Christophe

--
Christophe Leterrier
Researcher
Axonal Domains Architecture Team
CRN2M CNRS UMR 7286
Aix Marseille University, France

[Rosemary.White]

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Not sure if this will apply to animal tissues, but for a couple of plant
tissues where we kept getting background we blocked with pectin (started
with pectin for jam - too many other ingredients, but several purified
pectins are very cheap), and have also blocked with starch in the past (it
was instant mashed potatoes). But the comments about miscellaneous binding
to positive charges do resonate & will think about this in future.

cheers,
Rosemary

Dr Rosemary White
CSIRO Plant Industry
GPO Box 1600
Canberra, ACT 2601
Australia

T 61 2 6246 5475
F 61 2 6246 5334
E [hidden email]


On 21/03/13 8:23 PM, "Christophe Leterrier"
<[hidden email]> wrote:

>*****
>To join, leave or search the confocal microscopy listserv, go to:
>http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>*****
>
>Hi,
>
>Not a confocal question, but a lot of people are doing immuno here so I
>thought I'd ask. We are currently using gelatin as the main blocker in our
>imunocytochemistry protocol. I was wondering if we could get better
>staining (less background / less non specific labeling) by switching to
>something else and found Life Technologie's ImageIT FX Enhancer (
>http://products.invitrogen.com/ivgn/product/I36933). Does someone have
>experience with this, and is the added cost justified? I try to stay clear
>of proprietary formulas but sometimes (see Prolong Gold) a "secret"
>product
>does work better.
>
>Thanks for the advices,
>
>Christophe
>
>--
>Christophe Leterrier
>Researcher
>Axonal Domains Architecture Team
>CRN2M CNRS UMR 7286
>Aix Marseille University, France

[Lloyd Donaldson]

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If I remember correctly acetylated BSA is supposed to counteract this binding to +ve charges as well as acting as a blocking agent. We find it works well with plant material. No need for a separate blocking step just put it in with the antibody. Has anyone used this approach with animal cells ?


Dr Lloyd Donaldson
Project Leader - Microscopy & Wood Identification
Senior Scientist - Plant Cell Walls & Biomaterials
Scion - Forests, Products, Innovation
49 Sala Street, Rotorua 3010
New Zealand
Ph 07 343 5581
www.scionresearch.com




-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of [hidden email]
Sent: Friday, 22 March 2013 10:39 a.m.
To: [hidden email]
Subject: Re: Blocking in immunocytochemsitry - ImageIT FX Enhancer?

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
*****

Not sure if this will apply to animal tissues, but for a couple of plant tissues where we kept getting background we blocked with pectin (started with pectin for jam - too many other ingredients, but several purified pectins are very cheap), and have also blocked with starch in the past (it was instant mashed potatoes). But the comments about miscellaneous binding to positive charges do resonate & will think about this in future.

cheers,
Rosemary

Dr Rosemary White
CSIRO Plant Industry
GPO Box 1600
Canberra, ACT 2601
Australia

T 61 2 6246 5475
F 61 2 6246 5334
E [hidden email]


On 21/03/13 8:23 PM, "Christophe Leterrier"
<[hidden email]> wrote:

>*****
>To join, leave or search the confocal microscopy listserv, go to:
>http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>*****
>
>Hi,
>
>Not a confocal question, but a lot of people are doing immuno here so I
>thought I'd ask. We are currently using gelatin as the main blocker in
>our imunocytochemistry protocol. I was wondering if we could get better
>staining (less background / less non specific labeling) by switching to
>something else and found Life Technologie's ImageIT FX Enhancer (
>http://products.invitrogen.com/ivgn/product/I36933). Does someone have
>experience with this, and is the added cost justified? I try to stay
>clear of proprietary formulas but sometimes (see Prolong Gold) a "secret"
>product
>does work better.
>
>Thanks for the advices,
>
>Christophe
>
>--
>Christophe Leterrier
>Researcher
>Axonal Domains Architecture Team
>CRN2M CNRS UMR 7286
>Aix Marseille University, France

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[Phillips, Thomas E.]

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Jason - Did you accidently invert the charges in your reply - according to Invitrogen's website "Alexa Fluor(r) dyes and many other dyes have positively charged modifications..." and usually when one sees high background binding to the nucleus, it is assumed the negatively charged nucleic acids are the culprit. Tom


Thomas E. Phillips, Ph.D
Professor of Biological Sciences
Director, Molecular Cytology Core
2 Tucker Hall
University of Missouri
Columbia, MO 65211-7400
573-882-4712 (office)
573-882-0123 (fax)
[hidden email]

http://biology.missouri.edu/people/?person=88
http://www.biotech.missouri.edu/mcc/


-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Kilgore, Jason
Sent: Thursday, March 21, 2013 11:18 AM
To: [hidden email]
Subject: Re: Blocking in immunocytochemsitry - ImageIT FX Enhancer? **vendor reply**

*****
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** Vendor Reply **

Hi, Christophe,

I actually helped develop that product.  The Image-iT FX Signal Enhancer solution isn't meant to replace protein blocking, and won't work as well as a substitute.  Most dyes have a negative charge, which makes them nonspecifically attracted to positively-charged areas on cells and tissues (in cultured cells, that's mainly the nuclei and mitochondria.  Brain tissue cryosections, it's the myelin).  The Signal Enhancer blocks the positive charges.  But you should still do a subsequent protein blocking.  Not everyone needs it, but if you see background with a secondary-only control that includes a protein blocker, then you might want to add it as a step.  I got to where I used it with every ICC / IHC protocol.

I've never found gelatin to be very effective in my assays as a protein blocker.  I generally recommend 6% Bovine Serum Albumin / 10% normal serum / PBS.  Another commercial option (one of those "proprietary" things you avoid) is called BlockAid, from Life Technologies, which is a mix of various protein blockers.  Use it undiluted or lightly diluted as a blocker and in with your primary.  I've found it to be as good or better than any other common blocking option I've tried for ICC / IHC (even though we market it for blocking microspheres).

Cheers,

Jason

Jason A. Kilgore
Technical Application Scientist
Molecular Probes Labeling and Detection Technologies Cells Systems Division
 
T 1 800 955 6288 then option 4, then option 6,  or  541 335 0353 * F 541 335 0238
29851 Willow Creek Rd * Eugene * OR * 97402-9132 * United States www.invitrogen.com/technicalsupport


-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Christophe Leterrier
Sent: Thursday, March 21, 2013 2:24 AM
To: [hidden email]
Subject: Blocking in immunocytochemsitry - ImageIT FX Enhancer?

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
*****

Hi,

Not a confocal question, but a lot of people are doing immuno here so I thought I'd ask. We are currently using gelatin as the main blocker in our imunocytochemistry protocol. I was wondering if we could get better staining (less background / less non specific labeling) by switching to something else and found Life Technologie's ImageIT FX Enhancer ( http://products.invitrogen.com/ivgn/product/I36933). Does someone have experience with this, and is the added cost justified? I try to stay clear of proprietary formulas but sometimes (see Prolong Gold) a "secret" product does work better.

Thanks for the advices,

Christophe

--
Christophe Leterrier
Researcher
Axonal Domains Architecture Team
CRN2M CNRS UMR 7286
Aix Marseille University, France

[Kilgore, Jason-2]

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Hi, Thomas,

No, the Alexa Fluor dyes and most others have a net negative charge (consider, for example, Alexa Fluor 488: http://probes.invitrogen.com/media/structure/3630.jpg), but what the web site refers to are special modifications of those dyes to add positively-charged functional groups (like amines).

It is thought that the positive charges in the nuclei that attract the dyes may be due to histones, but we don't know for certain what it is in the nuclei that attracts the negatively-charged dyes to the nucleus.

Jason


-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Phillips, Thomas E.
Sent: Thursday, March 21, 2013 4:22 PM
To: [hidden email]
Subject: Re: Blocking in immunocytochemsitry - ImageIT FX Enhancer? **vendor reply**

*****
To join, leave or search the confocal microscopy listserv, go to:
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*****

Jason - Did you accidently invert the charges in your reply - according to Invitrogen's website "Alexa Fluor(r) dyes and many other dyes have positively charged modifications..." and usually when one sees high background binding to the nucleus, it is assumed the negatively charged nucleic acids are the culprit. Tom


Thomas E. Phillips, Ph.D
Professor of Biological Sciences
Director, Molecular Cytology Core
2 Tucker Hall
University of Missouri
Columbia, MO 65211-7400
573-882-4712 (office)
573-882-0123 (fax)
[hidden email]

http://biology.missouri.edu/people/?person=88
http://www.biotech.missouri.edu/mcc/


-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Kilgore, Jason
Sent: Thursday, March 21, 2013 11:18 AM
To: [hidden email]
Subject: Re: Blocking in immunocytochemsitry - ImageIT FX Enhancer? **vendor reply**

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
*****

** Vendor Reply **

Hi, Christophe,

I actually helped develop that product.  The Image-iT FX Signal Enhancer solution isn't meant to replace protein blocking, and won't work as well as a substitute.  Most dyes have a negative charge, which makes them nonspecifically attracted to positively-charged areas on cells and tissues (in cultured cells, that's mainly the nuclei and mitochondria.  Brain tissue cryosections, it's the myelin).  The Signal Enhancer blocks the positive charges.  But you should still do a subsequent protein blocking.  Not everyone needs it, but if you see background with a secondary-only control that includes a protein blocker, then you might want to add it as a step.  I got to where I used it with every ICC / IHC protocol.

I've never found gelatin to be very effective in my assays as a protein blocker.  I generally recommend 6% Bovine Serum Albumin / 10% normal serum / PBS.  Another commercial option (one of those "proprietary" things you avoid) is called BlockAid, from Life Technologies, which is a mix of various protein blockers.  Use it undiluted or lightly diluted as a blocker and in with your primary.  I've found it to be as good or better than any other common blocking option I've tried for ICC / IHC (even though we market it for blocking microspheres).

Cheers,

Jason

Jason A. Kilgore
Technical Application Scientist
Molecular Probes Labeling and Detection Technologies Cells Systems Division
 
T 1 800 955 6288 then option 4, then option 6,  or  541 335 0353 * F 541 335 0238
29851 Willow Creek Rd * Eugene * OR * 97402-9132 * United States www.invitrogen.com/technicalsupport


-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Christophe Leterrier
Sent: Thursday, March 21, 2013 2:24 AM
To: [hidden email]
Subject: Blocking in immunocytochemsitry - ImageIT FX Enhancer?

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
*****

Hi,

Not a confocal question, but a lot of people are doing immuno here so I thought I'd ask. We are currently using gelatin as the main blocker in our imunocytochemistry protocol. I was wondering if we could get better staining (less background / less non specific labeling) by switching to something else and found Life Technologie's ImageIT FX Enhancer ( http://products.invitrogen.com/ivgn/product/I36933). Does someone have experience with this, and is the added cost justified? I try to stay clear of proprietary formulas but sometimes (see Prolong Gold) a "secret" product does work better.

Thanks for the advices,

Christophe

--
Christophe Leterrier
Researcher
Axonal Domains Architecture Team
CRN2M CNRS UMR 7286
Aix Marseille University, France