Re: [Histonet] Safranine o staining and Problem in tissues

From:Gayle Callis

I sent the SafO/fast green method privately. 

As for your soft tissues, it sounds more like overdehydration in alcohols
during processing, and possibly paraffin is too hot at infiltration, check
temperature - it should be at 60C max.  
What is your processing schedule in terms of time per change? 

As for decalcification, endpoint should be determined either chemically or
by some other reliable method.  We have a quick, but relatively accurate
method since the xray machine went to another university. It is a weight
loss/weight gain method, and will work with all decalcifying agents.  If
you wish, I will attach to you. 

Routinely, we use 10 - 15% formic acid made up from 88% stock formic acid
OR you can use buffered formic acid, as EDTA will extract proteoglycans
which can change tinctorial quality of cartilage staining. Buffered formic
acid can be made in lab using formic acid with  either sodium formate or
sodium citrate, these are available commercially. These recipes are found
in histotechnology books under bone decalcification chapters.  Buffered
formic acid contains approx 4.5% formic acid with the buffer salt and is
much slower to complete decalcification. 

Always make sure bones are fixed totally, rat joints can take as long as a
week to fix in NBF, and change fixative after a couple of days to insure
fixative is replenished.  Clean off skin and extra muscle without damaging
joints to facilitate fixation and decalcification.  Another good, but fast
decalcifier is 10% formic acid with 4% hydrochloric acid, and joints will
take only day or so to decalcify.  We prefer the slower, gentler 15% formic
acid, and do endpoint testing faithfully.  

I suggest you also do back in Histonet archives and read discussion on
decalcification as much of what is said here is already there, plus other
interesting commentary.
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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