[Histonet] Bouins, Brittling, and Blocks

[Saleem Babri]

Hello,
I learned of this listserve group at the recent meeting of the Biological Stain Commission.  I wanted to join immediately because I teach histology to undergraduate students. We work through the whole paraffin technique in lab using manual methods.  We are always coming up with some questions.  Here are a few:

1.  An  attendee at the Stain Commission meeting mentioned using Bouin's fixative  as a mordant prior to Mallory staining of formalin fixed tissues.  We have been using saturated mercuric chloride in 5% acetic acid.  Is Bouin's better?  Is it less toxic?  For how long should the sections be immersed in it?  Do you post-treat with lithium carbonate to remove the yellow picric acid before proceeding to the next step?

2.  How long can you store formalin fixed tissues in 70% alcohol?  Is it best to store them at room temperature or the refrigerator?

3.  Our mouse tissue blocks are coming out brittle, dry and hard to section with the exception of lung and testis samples.  I attributed this to incomplete paraffin infiltration, but lengthening the parafffin baths has not helped.  It was suggested to me that perhaps we are dehydrating too long.  We have been using 3 changes of a 100% alcohol for 1 hour each.  What would you recommend?

Many thanks in advance for your suggestions.
Elizabeth

Hello Elizabeth,
I read your email on the histonet post. l faced similar problems with peripheral nerves of human cadavers during my study. Please have a look at the following suggestions which I found helpful thanks to the technical staff in our histology lab in the Anatomy Department at the University of Queensland Australia.

1. Bouin's fixative is a very effective mordanting solution. For human cadaveric specimens that were embalmed six months prior to commencement of the study, nerve tissue was harvested and mordanted in Bouin's at 4C and continually agitated for a minimum of 24 hours. At the completion of 24 hours the picric acid was removed using saturated solution of lithium carbonate to facilitate proper staining. 

2. Literature has shown that you can store formaldehyde fixed tissue in alcohol for an indefinite period of time. Controversies do exist which indicate that the quality of the tissue is affected (becoming more brittle). It is always better to store them at 4C which can slow down the brittling of tissue preserved in alcohol.

3. Your sections might be hard because you might be using too high a concentration of formaldehyde/glutaraldehyde to fix the tissues. Moreover embedding could be a problem. Before you section the tissues might put them in the refrigerator for at least 15 minutes. Try using 80, 90, 100% alcohol for the three changes instead of 100% alcohol for all the three changes.