[Histonet] MacNeal's tetrachrome

[kbowden]

5% MC NEAL'S STAIN
from the lab of Dr. Robert Schenk
Bern Switzerland
Milled/Surface Stain

1.    Agitate in 50% EtOH    10 to 15 min.
2.    Distilled water wash    3 min.
3.    Agitate in 0.1% Formic acid    5 to 10 min.
4.    Distilled water wash    3 min.
5.    Distilled water wash    3 min.
6.    Distilled water wash    3 min.
7.    5% Mc Neal's (Filtered)    5 min.
8.    Distilled water wash    3 min.
9.    Distilled water wash    3 min.
10.    Distilled water wash    3 min.
11.    70% EtOH    1 dip
12.    0.1% Aqueous Basic Fuchsin (Filtered)    15 to 30 sec
13.    Distilled water wash    3 min.
14.    Distilled water wash    3 min.
15.    Distilled water wash    3 min.
16.    Air dry


5% Mc Neal's Stain

Mod. Mc Neal's Tetra chrome (KEEP IN DARK)    5 ml.
0.1% Toluidine Blue aqueous                                    2.5 ml.
Distilled Water                                                        
    42.5 ml.
   Stir on Stir plate, filter afterwards


0.1% Basic Fuchsin

Basic Fuchsin                                                            
0.5 gm
Deionized water                                                        
500.0 ml


0.1% Formic Acid

Formic Acid                                                            
   1.1 ml
Deionized water                                                        
1000.0 ml

10% Mod. Mc Neal's Tetrachrome (STOCK)

Methylene Blue Chloride (Merck 6045-color 52015)    1.0 grams
Azure A Eosinate (Chroma 1A 328)                            1.6 grams
Methyl Violet, Bernthsim (Chrome 1A 098)                    0.2 gram
Methyl Alcohol                                                        
    500 ml
Glycerin                                                                
        500 ml
   Heat at 50°C for 1 day, then 37°C for 3 days in oven (capped).  
Filter and store in dark bottle in dark.


Results:
    bone    - reddish/purple
    cells/nuclei    - blue
    Cartilage    - blue/purple
    ligamenti, connective tissue    - pinkish/red

--
Karen Bowden
Staff Research Associate II
University of CA, San Diego
Department of Orthopedics
9500 Gilman Dr. 0630
La Jolla, CA 92093-0630
858-534-4655 voice
858-534-5304 fax


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Orla Gallagher wrote:
> Dear Histonetters,
>
> Our lab would like to try staining some undecalcified LR White-
> embedded bone sections with MacNeal's tetrachrome stain. I've 
> found a recipe on the Histonet archive which mentions making up 
> the stain from scratch but doesn't say how long to stain for or how 
> to treat the sections after staining. The recipe also mentions  
> etching in formic acid before staining. Has anyone got some advice 
> as I haven't tried any etching techniques before?
>
> Many thanks,
> Orla
> ******************************
>
> Ms. Orla Gallagher
> Academic Unit of Bone Biology
> D Floor (DU20) Medical School
> Henry Wellcome Laboratories for Medical Research
> University of Sheffield
> Beech Hill Road
> Sheffield S10 2RX
> www.sheffield.ac.uk
>
> E-mail:o.m.gallagher <@t> sheffield.ac.uk
> Tel:   0114 271 3783(lab)
>        0114 271 3337(office)
> Fax:   0114 271 1711 
>
>
>
>
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