ADENOSINE TRIPHOSPHATASE (ATPASE)
互联网
SECTIONS
Unfixed cryostat or cold Formol calcuim/gum sucrose fixed on muscle biopsy sections.
SOLUTIONS
a. 0.1M Glycine buffer.
- 0.75g glycine + 0.585g NaC1 made up to 100ml with distilled water.
b. O.1M glycine/NaC1 buffer with 0.75M CaC12
- 50ml 0.1M glycine buffer
- 10ml 0.75M CaC12 -11.03g CaC12 2.H20/100ml.
- Add approx. 22ml 0.1M NaOH until 9.6-9.8 pH
METHOD At pH 9.4.
- Incubate freshly cut sections at 37C for 11 minutes, in the following
-
solution
- 5mg ATP. dissolved. Add 10ml of solution (b) adjust to pH 9.6-9.8.
- Rinse well in distilled water.
- Immerse in 2% COC12 for 5 minutes.
- Rinse well in distilled water + 3 or 4 in tap water.
- Immerse in dilute (1:10) ammonium sulphide solution for 30 seconds.
- Rinse well in running tap water.
- (optional) stain in Harris's haematoxylin, blue in tap water.
- Mount in glycerine jelly.
METHOD At pH 4.2 and 4.6
- Preincubate at 4C in 0.1M sodium acetate buffer with 10mM ETDA added (0.372g/100ml of buffer) for 10 minutes at pH 4.2 or 4.6.
- Wash in distilled water.
- Proceed as for the pH 9.4 method.
NOTES
Only the pH 4.2 sections need counterstaining in haematoxylin. Sections must be VERY well washed after steps 3 and 5 to remove any traces of previous solutions