[Histonet] Re: Histonet Digest, Vol 39, Issue 35

From:John PJ Coleman

1) Nail Fungus:  In our lab we use PAS with a light green  
counterstain. We soften them in "Nair" first. 10 min 1% periodic  
acid, rinse DIH2O, 20 min schiff reagent, Develop in warm standing  
tap water, 2% Alc Lt green solution 2-3 min, run down , cover.  
Charged slides so they don't wash off.

2) ER/PR- our docs get nervous if the control isn't at least 50%  
strongly pos. The best option is a mixed block with multiple cases of  
varying expression. Embed 6 slivers from 6 separate cases, document  
your "reactivity map" include liver or tonsil which should always be  
negative, and voila! multiple ranges and neg tissue control on one  

3) Microwave venting: Our microwave processors are vented to outside.  
If the stains you use in the microwave include AFB (Carbol  
fuchsin=phenolic) PAS (schiff= sulfite) or GMS (methenamine) then you  
may have to at least use them under a hood.

4) IHC/DAB- I've coverslipped  DAB IHC's after drying well with no  
ill effect, no alcohol or xylene.

5) Cheese brains- If your primary freeze produces no artefact,  
(cheese holes) then holding at a colder temp after adding OCT will  
not create them. Your sucrose treatment already prevents  big ice  
crystals from forming in the brain/producing cheese holes when you  
freeze in the cryostat. (Wood frogs, Rana sylvatica, hibernate  
completely frozen using this method, defrost in the spring and walk  
away with no cheesy brain holes) The faster the freeze, the smaller  
the crystal. We freeze in isopentane cooled with LN2, then add OCT,  
then store @ -70 and get no freeze cheese.

6) Recycler- Anatech sells buffer packs for recycled formalin, and  
FDA doesn't certify alcohol, xylene or formalin, but may restrict the  
use of such in FDA approved procedures, such as Herceptest or  
PathVysion, etc. Check the package inserts for your FDA approved  
tests to see if recycled stuff is allowed or not.   PS- recycling  
formalin= bad idea. The theory doesn't make sense to me. It isn't a  
solvent, formaldeyhde molecules are lost in fixation to the tissue,  
is't more pain than it's worth.  I liken it to distilling urine to  
save on distilled water costs. I'd much rather spend that time on  
making my hematoxylin. Now THAT's fun!

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