[Histonet] Mayer's Hematoxylin on frozen tissue

[Stacey Barrick]

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