[Histonet] Re: Oil Red O and Mayers

[gayle callis]

Stacey, 

 

You wrote:  You wrote:  

 

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.

 

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Possibilities here are:  

 

1.  Not getting rid of the isopropyl alcohol adequately.  Try a longer rinse
than 1 minute to rehydrate the section, and add an additional DI water rinse
to make sure the alcohol is removed

 

However there is a much better Oil Red O stain than the one you could be
using now (you didn't give method e.g. messy Oil Red O with propylene
glycol, and without isopropyl rinsing.    I am forwarding Churukians Oil Red
O under separate cover which has never failed on our frozen sections.  It is
easy to make up and use.   We also prefer Gill 2 hematoxylin staining which
takes less time and requires a mild bluing solution or you can simply use
warm water to blue the section.   If Gill 2 seems too dark, use Gill.   I
have the original Journal of Histotechnology publication by Churukian if you
want it.  We always do Oil Red O stain before counterstaining with
hematoxylin.  

 

2.   How fresh is your  Mayers?   It could be outdated or needs changing?


 

Also, after final tap water rinse, we coverslip without an extra deionized
water rinse.     

 

Good luck

 

Gayle Callis

HTL/HT/MT(ASCP)