[Histonet] Decal question

Gayle Callis gcallis <@t> montana.edu
Wed Apr 14 12:58:41 CDT 2004

There is a better way to determine endpoint than cutting with a scalpel.
You can do a weight loss, weight gain and if working with many mice, you
can do one or two samples which will be relatively the same in weight, age
of animal etc. and endpoint test these when doing batch decalcification. 

It is not the decalcification that causes bone to fall off slides -
problems with ACID decalcifiers show up as poor H&E staining. Your problem
would happen with EDTA decalcified bone also.  

Waterbath is a bit hot, use Tissue Prep 2 to infiltrate or at least embed
the bones in, harder paraffin and supports bone better.  Trim block face
and soak a bit on ice water (don't oversoak if bone swells out of block,
not good) on top of ice block, cut with a NEW knife blade, preferred is
high profile Dura Edge or EdgeRite (backwards naming??) from Richard Allan
for a sturdier blade.  If you want section to flatten, you can either place
it on 10% alcohol cold water bath, pick up on slide and go to warm bath but
don't let section float off slide, just let it stretch out in warm water.
You are breaking surface tension and allowing cartilage (soft) stretch out
in relation to the cortical bone (very hard).  The hotter the waterbath the
more likely the wrinkles will set permanently.  Do not flatten with heated
surface after picking up either, this can explode section.  

Have also used a 5% DMSO waterbath, glass insert in waterbath with 5% DMSO,
let this heat up and lay sections on this.  Caveat:  DMSO is not to touch
skin NOR be breathed, and it will cause metal parts to rust.  It works, did
a whole rabbit knee study with this.   Some people have used Tween 20 in
waterbath i.e. a detergent that allows section to flatten nicely.  

When you pick up on Plus charge, drain well, and then go to 37C - 40C
heating platform and allow the sections to dry FLAT.  We never dry at 60C,
as this causes overdrying of cartilage, it curls off slide during rinses
and do the latter very gently. Do not use ammonium hydroxide for bluing,
use Scotts tap water or Richard Allan bluing reagent. If you still have
problems you can presub clean, regular microscope slides with chrome
Gelatin but use 275 bloom gelatin, the larger gelatin molecule is superior
for holding boney things.  Dry flat at 40C, overnight, even longer is
better.  We have even put a few granules of this gelatin in the waterbath
as it is heating and use regular Superfrost (NOT positive charge) to pick
up section.  Don't overuse 275 bloom, it can cause unsightly background, or
presub, then dip slide in NBF 10 dips,  rinse and dry.   

Not knowing source of your positive charged slides, ours are made by Erie
under Fisherbrand name or VWR brand name as long as they say Plus Charge.  

Good Luck,

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

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