Sorry folks

I seem to have caused more confusion than usual with my sloppy cut and paste
of this method.  Here is a tidier version.  A further thought is that I have
only ever done this on human muscle.  So I hope I'm not sending you up a
blind alley!

If anyone still has problems email me and I will send you an attachment.

Andy Shand

Technical tips

1. The sections may be "held" in 1% calcium chloride for up to 10 minutes
before being stained.

Stock solutions

Acid pre-treatment
Calcium chloride (dihydrate) -     1.30g
Potassium acetate  -     2.45g
Distilled water   - 500mls
Adjust to pH 4.5 with concentrated acetic acid.



Triz buffer pH 7.8
Trizma base           6.05g
Calcium chloride (dihydrate)-1.3g
Distilled water  - 500mls
Adjust to pH 7.8 with 5M HCl.


1% Calcium chloride


0.18M Calcium chloride
 Calcium chloride -     2.646g
Distilled water  - 100mls


Incubation medium
Distilled water   - 15mls
Potassium chloride  - 0.0925g
ATP (disodium salt)  - 0.038g
Calcium chloride (0.18M) - 2.5mls
Sigma 221 buffer  - 1.675mls
Adjust to pH 9.4 with 1M or 5M HCl.

METHOD

1. Treat air dried frozen sections with acid pretreatment solution (6 mins.)
2. Transfer slides through three rinses in Triz buffer, 2 mins. each.
3. Place slides in incubation medium pH 9.4 for 25 mins. at room temp.
4. Rinse in three changes of 1% calcium chloride 5mins.
5. Drain and stain slides in 0.1% toluidine blue for 90 seconds.
6. Rinse  very quickly (no more than seconds) in distilled water to remove
excess stain.
7. Dehydrate rapidly (5 dips each of 95% alcohol and two changes of 100%
alcohol).
8. Clear in two changes of xylene and mount in DPX.

Results

Type I flibres  - Turquoise   Type IIA fibres - Light red
Type IIB fibres - Violet  Type IIC fibres - Blue