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

From:Gayle Callis

Mallory's triple stain (I really had to dig into my histopast from 
1962!)  The reason mercuric chloride is used as a mordant for this stain is 
that Zenkers (which contains mercuric chloride) was always a fixative of 
choice for this special stain.  One could actually fix in NBF, and mordant 
in Zenkers before staining , when we did this stain in the first 
histotechnics class.

I do not recall ever using Bouins as a mordant for the Triple, but John 
Kiernan may set us straight on this whole subject. We only used Bouins 
either as a fixative or mordant for Massons Trichrome, but if it works for 
Mallory's, I would do it instead of toxic mercuric chloride.

Overprocessing of your mouse tissue sounds like the culprit for your dry, 
brittle tissues.  Mouse tissues are generally very small and can be 
processed 30 to 45 min per change.  Liver and brain  must be totally fixed 
before processing due to their homogenous nature or processing finished the 
fixation and generally dries out the tissues even more.

70%, 80%, 95% x 2, 100% x 2, xylene X 2 with 3 changes of paraffin at 30 
min each as heat will dry the tissues out even more.

After trimming a block, set it on cold ice water block, but return it to 
the microtome and DO NOT TRIM OFF WHAT YOU HAVE JUST SOAKED!!!!  You want 
the first sections that come off the sharp blade.   If your room is really 
cold, you can even use a RT water soak, or try a gauze soaked in water over 
the just trimmed block.

We simply wash Bouins fixed or mordanted sections in running tap water 
until the color is gone, approx 5 min or so.

Gayle Callis
Research Histopathology Supervisor
Veterinary Molecular Biology
Montana State University - Bozeman
PO Box 173610
Bozeman MT 59717-3610
406 994-6367
406 994-4303 (FAX)

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