CTC staining protocol.pdf

SUBHASH C. JUNEJA, K.S., PH. D. CTC Stainig - Laboratory Protocol STOCK SOLUTIONS

1) 2M Tris-base

Dissolve 24.22 gm Tris-base in 100 ml distilled 2) Cysteine

Stock Solution (X10)

2M Tris-base

NaCl

water

10 ml

7.597

DL-Cysteine

gm

0.606 gm

Make Final Volume to 100 ml

3)

12.5% Glutaraldehyde in 1M Tris (pH 7.8) Add 5 ml of 2M Tris to 1.78 ml of 70% glutaraldehyde

Adjust pH 7.8,

Make final volume to 10 ml

WORKING SOLUTIONS

1) 0.625% glutaraldehyde

Dilute stock 12.5% glutaraldehyde 1:20 with distilled water.

(This solution can be kept in 4°C 2) CTC solution

Dissolve 2.6 mg CTC in 7 ml chillled distilled water, and add 1 ml of Cystiene stock solution,

Adjust pH 7.8,

Make final volume to 10 ml

Aliquot the stain into small fractions and freeze.

CTC is light sensitive, keep in aluminium wrapped tubes

METHOD;

SLIDE STAINING

* Transfer 5-10 ul of sperm suspension on to a slide (^cCaS^C

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* Add equal volume of CTC stain (5-10 ul), mix the sperm suspension with a glass rod.

* Add 3.5 ul of working glutaraldehyde (0.625%) onto the slidg followed by thorough mixing

* Cover with a cover slip sealed with a nail polisher.

* Keep the slide at 4 C in a dark container (slide folder).

* Score the CTC pattern on the same day U~^&U^ U\/ J&fkk: