[Histonet] decalcification[Scanned]

[Kemlo Rogerson]

Ergo, EDTA. If the main desire is to retain staining integrity, then
anything containing an acid will compromise that. Never tried using EDTA at
raised temperatures, that may increase the rate of decalcification; depends
on the size of the lump too.

-----Original Message-----
From: Gayle Callis [mailto:gcallis <@t> montana.edu] 
Sent: 13 January 2005 18:16
To: Bernardo Vargas-Angel; Histonet <@t> lists.utsouthwestern.edu
Subject: Re: [Histonet] decalcification[Scanned]

Bernardo,

If you control the decalcification using a decalcification endpoint test, 
you can prevent some loss but strong mineral acids still create some loss 
of positive hematoxylin staining.    If you prevent overexposure aka 
"overdecalcification" of your fixed calcified tissue to any acid, even 
formic acid - you can minimize your problem or acid protein hydrolysis will 
affect nuclear i.e. DNA and RNA staining.  I presume, from where you are 
working, that you are decalcifying coral, sea shells, etc??  Some have used 
Davidson's fixative which contains acetic acid and actually does some 
decalcification along with fixation.  This is a popular fixative for people 
in Fish and Wildlife here for their trout, fresh water fish studies.

There are ways to speed up decalcification other than just using a 
different acid decalcifier.   Acid, no matter which one, can still damage 
nuclear staining IF you don't know when your sample is free of calcium.

One can use a higher concentration of formic acid, gentler than HCl or 
nitric, but endpoint determination is still advisable as leaving samples in 
formic acid will result in the problem you are experiencing.   One can use 
lower concentration of HCl from 10% to 5% or less and it will still be 
fast. There is no law to say you can't dilute a commercial decalcifier (HCl 
variety) and usually around 10 - 12% HCl in stock solution.  Look at the 
MSDS and see what HCL concentration is in stock decalcifier and dilute 
accordingly.

Control is still key to avoiding nuclear staining loss.



At 10:16 AM 1/13/2005, you wrote:
>Could anyone out there suggest a method for rapid acid decalcification that
>does not interfere with basophilia?
>
>Bernardo Vargas-Angel  Ph.D.
>Research Scientist
>National Coral Reef Institute
>Nova Southeastern University
>8000 N Ocean Drive, Dania Beach, FL, 33004
>Voice +(954) 262-3677
>Fax +(954) 262-4027
>http://www.nova.edu/ocean/vargas-angel/index.html
>
>
>_______________________________________________
>Histonet mailing list
>Histonet <@t> lists.utsouthwestern.edu
>http://lists.utsouthwestern.edu/mailman/listinfo/histonet

Gayle Callis
MT,HT,HTL(ASCP)
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)



_______________________________________________
Histonet mailing list
Histonet <@t> lists.utsouthwestern.edu
http://lists.utsouthwestern.edu/mailman/listinfo/histonet