Please be aware that carry over of the glacial acetic acid 
can effect the Safranin O and the next thing you know, 
you'r not getting any staining.  I have substituted Gill's 
III for the Weigert's and still get very good staining.

Karen Bowden
kbowden@ucsd.edu
UCSD
Department of Orthopedics
La Jolla, CA


SAFRANIN O/FAST GREEN FOR PARAFFIN

1.    Xylene(x3)    10 min
2.    100% EtOH (x2)    3 min
3.    95% EtOH    3 min
4.    70% EtOH    3 min
5.    Weigert's Iron Hematoxylin    8 min
6.    Distilled water (x3)    3 min
7.    1:5000 Fast green    3 min
8.    1.0% Glacial acetic acid    3 dip
9.    1:50 Safranin O    6 min
10.    70% EtOH    1 min
11.    95% EtOH (x2)    1 min
12.    100% EtOH (x2)    1 min
13.    Xylene (x2)    1 min
14.    Mount with mounting media

WEIGERT'S IRON HEMATOXYLIN
     SOLUTION A
Hematoxylin crystals    1.0 gm
Alcohol, 95%    100.0 ml

     SOLUTION B
Ferric Chloride, 29% aqueous    4.0 ml
Distilled water    95.0 ml
Hydrochloric acid, concentrated    1.0 ml
     Equal parts of Solution A and Solution B.  Use within 
24 hours of combining.

SAFRANIN 0
Deionized water    10 ml
Safranin O    1 ml  of 1%

FAST GREEN
Deionized water    50 ml
Fast Green    1 ml of 1%

GLACIAL ACETIC ACID
Deionized water    990 ml
Glacial Acetic Acid    10 ml

Results:
     Nuclei    - black
     Cytoplasm    - gray green
     Mucus, Cartilage    - orange red

N.B.    If cytoplasm, muscle, collagen etc. do not stain 
green, reduce the time in safranin O

REFERENCE: Journal of Bone and Joint Surgery, Vol 53A, No. 
1 1971 pp69.

CYNTHIA HAYNES wrote:
> 
> 
> 
> 
> Hello my fellow histonetters,
>        Does any one have a protocol for Fast green(methyl 
> green)-Saffrain-o stain for paraffin on decaled bone. The procedure I am 
> using is not working for some strange reason. If any has a procedure for 
> making up the solutions I would greatly appreciated.
>                                      Thanks in Advance
>                                    Cynthia Haynes H.T.
> 
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