The procedure is as follows:

1) Whole mouse or mouse or rat pups are evicerated and any fur is removed.
2) Fix in ample volume of 95 % ethanol for 3-5 days depending on the size of
the specimen.
3) Place specimens into acetone (anhydrous) to remove fat. Change the
acetone periodically. When 5 - 10 % (by volume) of "spent" acetone is placed
into a graduated cyclinder containing 90 % (by volume) dH20 and remains
clear, the specimens are ready for further processing.
4) Stain in the following solution for 2 -3 days at 37 oC (loosely capped
container or covered with parafilm):
	0.3 % filtered alcian blue 8GS (CI # 74240) in 70 % ethanol -- 1
volume
	0.1 % filtered alizarin red s (CI # 58005) in 95 % ethanol -- 1
volume
	Glacial acetic acid -- 1 volume
	70 % ethanol -- 17 volumes
e.g. for 1000 mL volumes are 50 ml, 50 ml, 50 ml and 850 mL respectively
5) wash well in gently running water
6) clear in 1 % aqueous KOH for 12-48 hours or until skeletons are clearly
visible through the skin and surrounding tissue.
7) transfer specimens to a solution of 20 % glycerin in 1 % KOH for 1 - 5
days
8) transfer to a solution of 50 % glycerin (aq.) for 2- 5 days
9) transfer to a solution of 80 % glycerin (aq.) for 2 - 5 days
10 ) store in 100 % glycerin.

Results:
mineralized bone = red
cartilage = blue
tissues = clear

Reference: Minoru Inouye (1976) Cong. Anom. 16: 171-173. Differential
staining of cartilage and bone in fetal mouse skeleton by alcian blue and
alizarin red s.

I can email you a picture of the whole mice we did if you like. Good luck,
Donna Montague, Orthopaedic Research, UAMS Little ROck, AR

-----Original Message-----
From: Andrea Grantham [mailto:algranth@u.arizona.edu]
Sent: Tuesday, March 13, 2001 5:01 PM
To: histonet@pathology.swmed.edu
Subject: procedure staining for bone and cartilage


I was wondering if anybody does or has done a stain for bone and cartilage 
on mouse pups using Alizarin Red S and Alcian Blue. The investigator 
brought a great picture in a book and asked me to do this stain but I can't 
find a procedure.
Andi Grantham
.....................................................................
: Andrea Grantham, HT(ASCP)     Dept. of Cell Biology & Anatomy     :
: Sr. Research Specialist       University of Arizona               :
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:...................................................................:
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