Re: Rat vertebrate paraffin sections

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From:Barry Rittman <> (by way of histonet)
To:histonet <>
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                as you will be cutting paraffin sections at 50 microns,
I would recommend that you use a slightly softer wax (50-54 degrees) a
this will decrease shattering.
Also for staining thick sections, I prefer using progressive staining
with very dilute solutions of Ehrlich's hematoxylin, eosin etc. This
tends to prevent uneven staining. Can use 0.5% Hx in glass distilled
water overnight. This has the added advantage  of showing the individual
lamellae, areas of previously calcified cartilage etc.
You may want to try alcian blue staining for the cartilage as this can
also be used even on entire animals.
In fact no matter how thick your sections, you can always try surface
staining and then examine them by transmitted or reflected light.
To prevent cracking in the clearing steps I would recommend using
terpineol  (oil of lilacin) as a clearing agent instead of xylene or
xylene substitutes. The sections will remain supple in this agent.
Terpineol is however expensive and when mounting from terpineol you will
need to blot the sections with lens paper backed with filter paper to
remove the excess terpineol.
Call me if you need any more details.
Say hi to everyone in Iowa we spent many happy years there.
Barry wrote:

> Date:  12/10/1998  03:46 pm  (Thursday)
> From:  Clover Daley
> To:  pcu_dom.api:""
> Subject:  Rat vertebrate paraffin sections
> Dear Histonet followers:
> I am working on a protocol for processing zygapophyseal
> joints from L3-S1 of the rat vertebrate. The sections are to
> demonstrate any bridging of articular cartilage in these areas.
> The protocol that I have reviewed is similar to the one in the
> third edition of the AFIP manual, pg. 47-52 for processing
> human temporal bones.
> I would like to process in paraffin after decalcification instead
> of nitrocellulose.
> Questions:
> The procedure requires me to serial section each joint at 50
> microns and stain every tenth one in Ehrlich' Hematoxylin
> with a light green counterstain. Could I substitute Harris
> Hematoxylin?
> Does anyone have information on staining times for sections
> this thick?
> Also, if anyone has done staining to demonstrate cartilage
> bridging different from this, please let me know. I am open for
> any suggestions.
> Thank you,
> Clover Daley
> Palmer Center for Chiropractic Research
> Davenport, Iowa

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