[Histonet] Mayer's Hematoxylin on frozen tissue

[Rene J Buesa]

Mayer's hemaotoxylin (a "progressive" hematoxylin) is not adequate for FS.
Try Harris hematoxylin BUT stain first with the hematoxylin and with ORO after wards to obtain better results.
René J.

From: Stacey Barrick <barrickstacey <@t> yahoo.com>
To: "Histonet <@t> lists.utsouthwestern.edu" <Histonet <@t> lists.utsouthwestern.edu> 
Sent: Wednesday, February 27, 2013 11:20 AM
Subject: [Histonet] Mayer's Hematoxylin on frozen tissue

Hi everyone, 

We are trying to stain frozen cut sections of aorta with Mayer's Hematoxylin following Oil Red O staining. We cannot get hematoxylin staining to work. We are mainly seeing blue background but not labeling of nuclei.
Tissue is fixed with 4% PFA prior to sectioning. After tissue is cut; sections  are stained with Oil Red O
·         ddH2O
2’
·         60%
isopropanol 30s
·         Oil
Red O 18’
·         60%
isopropanol 30s
·         2x
ddH2O 1’

Sections are then stained with Sigma Mayer's Hematoxylin 
·    Rinse in deionized water

·   Stain in Mayer's Hematoxylin 1-5 min

·  Rinse in running tap water until nuclei are blue
·  Rinse in deionized water


We have tried staining in Hematoxylin for 3 min up to 15 mins
We have also tried rinsing 1 min up to 15 mins (checking at for staining at various timepoints) and always see the same result -some blue staining but no clear nuclear staining.

Does anyone have any suggestions as to why this isn't working?
Thanks!!

Stacey
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