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red corpuscles autofluorescence and nuclear endothelial cells staining

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Moreto Elias, Jemina Moreto Elias, Jemina
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red corpuscles autofluorescence and nuclear endothelial cells staining

Dear all,

 

I have two technical questions and I was wondering if you could have the solution.

 

1-       Do you know a way to avoid the presence of red blood corpuscle in tissues before performing immunofluorescence staining? Or otherwise, is there any way to avoid their autofluorescence?

 

2-       We are trying to stain the nucleus of endothelial retina vessels. We have tried TX100 permeabilization, as well as HCl with DMSO without success… DAPI is staining all kind of nucleus in this tissue but not endothelial cell ones.

 

Thank you in advance

 

Jemina Moretó Elias, PhD.

 

Confocal Microscopy

Cancer Epigenetics and Biology Program (PEBC)

IDIBELL, Catalan Institute of Oncology (ICO)

 

Avinguda Gran Via de l'Hospitalet, 199-203

08907, L'Hospitalet de Llobregat (Barcelona)

Tel. +34 932607500 ext 3176

 

 

Jurkevic, Aleksandr Jurkevic, Aleksandr
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Re: red corpuscles autofluorescence and nuclear endothelial cells staining

Jemina,

 

Regarding your first question, you need to perfuse a whole animal or organ, if possible, with 0.1M phosphate buffer (PB). Addition of some heparin and 0.1% sodium nitrite will help to  remove all erythrocytes even from smallest blood vessels.

 

Alexander Jurkevic

 

From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Moreto Elias, Jemina
Sent: Wednesday, June 16, 2010 6:38 AM
To: [hidden email]
Subject: red corpuscles autofluorescence and nuclear endothelial cells staining

 

Dear all,

 

I have two technical questions and I was wondering if you could have the solution.

 

1-     Do you know a way to avoid the presence of red blood corpuscle in tissues before performing immunofluorescence staining? Or otherwise, is there any way to avoid their autofluorescence?

 

2-     We are trying to stain the nucleus of endothelial retina vessels. We have tried TX100 permeabilization, as well as HCl with DMSO without success… DAPI is staining all kind of nucleus in this tissue but not endothelial cell ones.

 

Thank you in advance

 

Jemina Moretó Elias, PhD.

 

Confocal Microscopy

Cancer Epigenetics and Biology Program (PEBC)

IDIBELL, Catalan Institute of Oncology (ICO)

 

Avinguda Gran Via de l'Hospitalet, 199-203

08907, L'Hospitalet de Llobregat (Barcelona)

Tel. +34 932607500 ext 3176

 

 

Leigh Silvester Leigh Silvester
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Re: red corpuscles autofluorescence and nuclear endothelial cells staining

In reply to this post by Moreto Elias, Jemina
You could try using a red blood cell lysis buffer.

Protocol provided by most manufacturers is for whole blood, although this can be adapted for tissue.
Would need to wash tissue thoroughly afterwards to prevent deterioration of tissue and wash away any haemoglobin.

A typical blood protocol would be

Dilute one volume of cell suspension with 5-10 volumes of lysis buffer (155 mM NH4Cl; 10 mM KHCO3 and 0.1 mM EDTA) and incubate for 5 minutes at room temperature

This has been done on small tissue chunks satisfactorily.

Regards

Leigh Silvester


From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Moreto Elias, Jemina
Sent: 16 June 2010 12:38
To: [hidden email]
Subject: red corpuscles autofluorescence and nuclear endothelial cells staining

Dear all,

I have two technical questions and I was wondering if you could have the solution.

1- Do you know a way to avoid the presence of red blood corpuscle in tissues before performing immunofluorescence staining? Or otherwise, is there any way to avoid their autofluorescence?

2- We are trying to stain the nucleus of endothelial retina vessels. We have tried TX100 permeabilization, as well as HCl with DMSO without success... DAPI is staining all kind of nucleus in this tissue but not endothelial cell ones.

Thank you in advance

Jemina Moretó Elias, PhD.
 
Confocal Microscopy
Cancer Epigenetics and Biology Program (PEBC)
IDIBELL, Catalan Institute of Oncology (ICO)
 
Avinguda Gran Via de l'Hospitalet, 199-203
08907, L'Hospitalet de Llobregat (Barcelona)
Tel. +34 932607500 ext 3176
 
[hidden email]
www.pebc.cat

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