off topic: lignin staining

[Leigh Silvester]

Hi

A colleague is trying to stain some wood material for lignin using
phloroglucinol-HCl.

Due to the nature of the material it is currently being set in HEMA -
hydroxyethylmethacrylate.

Problem is that the stain does not appear to be compatible with this
resin.
The stain works fine on lose preparations. He is also aware of the light
sensitivity issue.

Any one suggest any pretreatments for the HEMA sections?
Perhaps there is an alternative lignin stain that is compatible with
this resin?

We have tried other resins but so far the HEMA one gives the best
sections with this material.

Any suggestions are very welcome.

Regards

Leigh Silvester
==================================================================
Division of Animal Sciences
University of Nottingham
Sutton Bonington Campus
Loughborough
Leicestershire
LE12 5RD
T: [phone]
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E: [hidden email]

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[Dale Callaham]

Leigh,

This is just a suggestion - I don't work with HEMA embedments, but I
have used the stain with material in LRWhite. The pararosanaline-based
Schiff's Reagent will stain the free amines of lignin and there are
typically few interferences in "acetic-alcohol" fixed plant tissues. It
is both colored and highly fluorescent with a Rhodamine exciter set. The
fluoresence is very stable and resistant to bleaching.

See the image on our web page - secondary wall thickenings of Plantago
xylem.
http://www.bio.umass.edu/microscopy.

Dale


Leigh Silvester wrote:

> Hi
>
> A colleague is trying to stain some wood material for lignin using
> phloroglucinol-HCl.
>
> Due to the nature of the material it is currently being set in HEMA -
> hydroxyethylmethacrylate.
>
> Problem is that the stain does not appear to be compatible with this
> resin.
> The stain works fine on lose preparations. He is also aware of the light
> sensitivity issue.
>
> Any one suggest any pretreatments for the HEMA sections?
> Perhaps there is an alternative lignin stain that is compatible with
> this resin?
>
> We have tried other resins but so far the HEMA one gives the best
> sections with this material.
>
> Any suggestions are very welcome.
>
> Regards
>
> Leigh Silvester
> ==================================================================
> Division of Animal Sciences
> University of Nottingham
> Sutton Bonington Campus
> Loughborough
> Leicestershire
> LE12 5RD
> T: [phone]
> F: [phone]
> E: [hidden email]
>
> NOTE - NEW TELEPHONE NUMBER
> ==================================================================
> Do you need to print this email?
> ==================================================================
>
> This message has been checked for viruses but the contents of an attachment
> may still contain software viruses which could damage your computer system:
> you are advised to perform your own checks. Email communications with the
> University of Nottingham may be monitored as permitted by UK legislation.

[Leigh Silvester]

Dale

Many thanks for this very promising response.

I should have mentioned that this material is going to be imaged by
light microscopy and not fluorescence/confocal.

Leigh

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]]
On Behalf Of Dale Callaham
Sent: 22 January 2010 11:43
To: [hidden email]
Subject: Re: off topic: lignin staining

Leigh,

This is just a suggestion - I don't work with HEMA embedments, but I
have used the stain with material in LRWhite. The pararosanaline-based
Schiff's Reagent will stain the free amines of lignin and there are
typically few interferences in "acetic-alcohol" fixed plant tissues. It
is both colored and highly fluorescent with a Rhodamine exciter set. The

fluoresence is very stable and resistant to bleaching.

See the image on our web page - secondary wall thickenings of Plantago
xylem.
http://www.bio.umass.edu/microscopy.

Dale


Leigh Silvester wrote:

> Hi
>
> A colleague is trying to stain some wood material for lignin using
> phloroglucinol-HCl.
>
> Due to the nature of the material it is currently being set in HEMA -
> hydroxyethylmethacrylate.
>
> Problem is that the stain does not appear to be compatible with this
> resin.
> The stain works fine on lose preparations. He is also aware of the

light

> sensitivity issue.
>
> Any one suggest any pretreatments for the HEMA sections?
> Perhaps there is an alternative lignin stain that is compatible with
> this resin?
>
> We have tried other resins but so far the HEMA one gives the best
> sections with this material.
>
> Any suggestions are very welcome.
>
> Regards
>
> Leigh Silvester
> ==================================================================
> Division of Animal Sciences
> University of Nottingham
> Sutton Bonington Campus
> Loughborough
> Leicestershire
> LE12 5RD
> T: [phone]
> F: [phone]
> E: [hidden email]
>
> NOTE - NEW TELEPHONE NUMBER
> ==================================================================
> Do you need to print this email?
> ==================================================================
>
> This message has been checked for viruses but the contents of an

attachment
> may still contain software viruses which could damage your computer
system:
> you are advised to perform your own checks. Email communications with
the
> University of Nottingham may be monitored as permitted by UK
legislation.

[Lloyd Donaldson]

Leigh

There is no really specific stain for lignin for either fluorescence or brightfield. For fluorescence we find autofluorescence to be the most reliable. Stains often give varying responses depending on whether the material is embedded or not, and what its embedded in.
However if you are using unembedded material then phloroglucinol will be good if it is a gymnosperm. I see you are working on Plantago so the Maule reagent is likely to be better.
For embedding try LR White hard formulation or SPurr resin. Spurr resin can be removed after sectioning which might improve staining. Try silane coated slides if you have adhesion problems.


Dr Lloyd Donaldson
Senior Scientist
Scion - Next Generation Biomaterials
49 Sala St. Rotorua
Private Bag 3020, Rotorua 3046
NEW ZEALAND
Ph: 64 7 343 5581
Fx: 64 7 343 5507
www.scionresearch.com



-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Dale Callaham
Sent: Saturday, 23 January 2010 12:43 a.m.
To: [hidden email]
Subject: Re: off topic: lignin staining

Leigh,

This is just a suggestion - I don't work with HEMA embedments, but I
have used the stain with material in LRWhite. The pararosanaline-based
Schiff's Reagent will stain the free amines of lignin and there are
typically few interferences in "acetic-alcohol" fixed plant tissues. It
is both colored and highly fluorescent with a Rhodamine exciter set. The
fluoresence is very stable and resistant to bleaching.

See the image on our web page - secondary wall thickenings of Plantago
xylem.
http://www.bio.umass.edu/microscopy.

Dale


Leigh Silvester wrote:

> Hi
>
> A colleague is trying to stain some wood material for lignin using
> phloroglucinol-HCl.
>
> Due to the nature of the material it is currently being set in HEMA -
> hydroxyethylmethacrylate.
>
> Problem is that the stain does not appear to be compatible with this
> resin.
> The stain works fine on lose preparations. He is also aware of the light
> sensitivity issue.
>
> Any one suggest any pretreatments for the HEMA sections?
> Perhaps there is an alternative lignin stain that is compatible with
> this resin?
>
> We have tried other resins but so far the HEMA one gives the best
> sections with this material.
>
> Any suggestions are very welcome.
>
> Regards
>
> Leigh Silvester
> ==================================================================
> Division of Animal Sciences
> University of Nottingham
> Sutton Bonington Campus
> Loughborough
> Leicestershire
> LE12 5RD
> T: [phone]
> F: [phone]
> E: [hidden email]
>
> NOTE - NEW TELEPHONE NUMBER
> ==================================================================
> Do you need to print this email?
> ==================================================================
>
> This message has been checked for viruses but the contents of an attachment
> may still contain software viruses which could damage your computer system:
> you are advised to perform your own checks. Email communications with the
> University of Nottingham may be monitored as permitted by UK legislation.

Disclaimer: This e-mail and any attachments may contain information which is confidential or subject to copyright. If you receive this e-mail in error, please delete it.
Scion does not accept responsibility for anything in this e-mail which is not provided in the  course of Scion's usual business or for any computer virus, data corruption, interference or delay arising from this e-mail.

[Cameron Nowell]

Hi List,
 
I am sure i found at some time in the past a table showing the realtive brightness of the AlexaFluors to each other. I have done some googling and haven't found it.
 
Am i going mad or does this list exist somewhere?
 
 
Cheers
 

Cam
 
 
 
Cameron J. Nowell
Microscpy Manager
Central Resource for Advanced Microscopy
Ludwig Insttue for Cancer Research
PO Box 2008
Royal Melbourne Hospital
Victoria, 3050
AUSTRALIA
 
Office: [phone]
Mobile: [phone]
Fax: [phone]
 
http://www.ludwig.edu.au/branch/research/platform/microscopy.htm
 

[Johnson, Iain-2]

http://www.invitrogen.com/site/us/en/home/References/Molecular-Probes-The-Handbook/Fluorophores-and-Their-Amine-Reactive-Derivatives/Alexa-Fluor-Dyes-Spanning-the-Visible-and-Infrared-Spectrum.html#datatable


Peak extinction coefficients (EC) in the body of the table, quantum yields (QY) in the footnotes.  "Brightness" = EC*QY.   Usual disclaimers apply relating to the fact that the QY is context dependent and the values in the table footnotes are for one particular context (free dye in PBS pH 7.2).

Iain



-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Cameron Nowell
Sent: Saturday, January 23, 2010 7:25 PM
To: [hidden email]
Subject: AlexaFluor Relative Brightness

Hi List,
 
I am sure i found at some time in the past a table showing the realtive brightness of the AlexaFluors to each other. I have done some googling and haven't found it.
 
Am i going mad or does this list exist somewhere?
 
 
Cheers
 

Cam
 
 
 
Cameron J. Nowell
Microscpy Manager
Central Resource for Advanced Microscopy
Ludwig Insttue for Cancer Research
PO Box 2008
Royal Melbourne Hospital
Victoria, 3050
AUSTRALIA
 
Office: [phone]
Mobile: [phone]
Fax: [phone]
 
http://www.ludwig.edu.au/branch/research/platform/microscopy.htm
 

[Cameron Nowell]

Hi Iain,
 
Thanks.
 
Does anyone out there know the QY of Alexa 405? It isn't in the table
 
 
Cheers
 
Cam
 
 

________________________________

From: Confocal Microscopy List on behalf of Johnson, Iain
Sent: Tue 26/01/2010 4:50 AM
To: [hidden email]
Subject: Re: AlexaFluor Relative Brightness



http://www.invitrogen.com/site/us/en/home/References/Molecular-Probes-The-Handbook/Fluorophores-and-Their-Amine-Reactive-Derivatives/Alexa-Fluor-Dyes-Spanning-the-Visible-and-Infrared-Spectrum.html#datatable


Peak extinction coefficients (EC) in the body of the table, quantum yields (QY) in the footnotes.  "Brightness" = EC*QY.   Usual disclaimers apply relating to the fact that the QY is context dependent and the values in the table footnotes are for one particular context (free dye in PBS pH 7.2).

Iain



-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Cameron Nowell
Sent: Saturday, January 23, 2010 7:25 PM
To: [hidden email]
Subject: AlexaFluor Relative Brightness

Hi List,

I am sure i found at some time in the past a table showing the realtive brightness of the AlexaFluors to each other. I have done some googling and haven't found it.

Am i going mad or does this list exist somewhere?


Cheers


Cam



Cameron J. Nowell
Microscpy Manager
Central Resource for Advanced Microscopy
Ludwig Insttue for Cancer Research
PO Box 2008
Royal Melbourne Hospital
Victoria, 3050
AUSTRALIA

Office: [phone]
Mobile: [phone]
Fax: [phone]

http://www.ludwig.edu.au/branch/research/platform/microscopy.htm

[Johnson, Iain-2]

Alexa Fluor 405 should have the same quantum yield as Cascade Blue (QY = 0.54 in water) since it is essentially the same molecule by a different name.

Iain

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Cameron Nowell
Sent: Monday, January 25, 2010 1:45 PM
To: [hidden email]
Subject: Re: AlexaFluor Relative Brightness

Hi Iain,
 
Thanks.
 
Does anyone out there know the QY of Alexa 405? It isn't in the table
 
 
Cheers
 
Cam
 
 

________________________________

From: Confocal Microscopy List on behalf of Johnson, Iain
Sent: Tue 26/01/2010 4:50 AM
To: [hidden email]
Subject: Re: AlexaFluor Relative Brightness



http://www.invitrogen.com/site/us/en/home/References/Molecular-Probes-The-Handbook/Fluorophores-and-Their-Amine-Reactive-Derivatives/Alexa-Fluor-Dyes-Spanning-the-Visible-and-Infrared-Spectrum.html#datatable


Peak extinction coefficients (EC) in the body of the table, quantum yields (QY) in the footnotes.  "Brightness" = EC*QY.   Usual disclaimers apply relating to the fact that the QY is context dependent and the values in the table footnotes are for one particular context (free dye in PBS pH 7.2).

Iain



-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Cameron Nowell
Sent: Saturday, January 23, 2010 7:25 PM
To: [hidden email]
Subject: AlexaFluor Relative Brightness

Hi List,

I am sure i found at some time in the past a table showing the realtive brightness of the AlexaFluors to each other. I have done some googling and haven't found it.

Am i going mad or does this list exist somewhere?


Cheers


Cam



Cameron J. Nowell
Microscpy Manager
Central Resource for Advanced Microscopy
Ludwig Insttue for Cancer Research
PO Box 2008
Royal Melbourne Hospital
Victoria, 3050
AUSTRALIA

Office: [phone]
Mobile: [phone]
Fax: [phone]

http://www.ludwig.edu.au/branch/research/platform/microscopy.htm