[Histonet] mordanting in Bouin's, storage in 70%, processing mouse tissues

[Geoff McAuliffe]

Hi Elizabeth:

Wurdak, Elizabeth wrote:

>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?
>  
>

Mercuric cholride is very toxic and polutes the environment. Even the 
water and alcohol washes after HgCl should not go down the drain. One 
does not need to mordant in Bouin's fix, one half or 3/4 saturated 
picric acid will do nicely. Treat blocks at room temp overnight, wash in 
water. I think you are better off treating the blocks rather than the 
sections but I do not have evidence for this idea, just the idea of 
preserving things before hitting them with alcohol. I post-treat the 
sections with LiCO3, much faster than treating the whole block. I 
suspect that any carbonate will do for this? If your safety people don't 
want you to have a bottle of picric acid in the lab, just buy some sat. 
aqueous solution.

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

You can store it as long as you want to. However. long storage does 
extract cytoplasm and will decrease staining. How long is long? I don't 
know.

>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?
>  
>

Sounds like over-processing to me as well.

Geoff

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Geoff McAuliffe, Ph.D.
Neuroscience and Cell Biology
Robert Wood Johnson Medical School
675 Hoes Lane, Piscataway, NJ 08854
voice: (732)-235-4583; fax: -4029 
mcauliff <@t> umdnj.edu
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