Sucrose Spreading of Mammalian Spermatocytes

1. Decapsulate Testes and place in bubble of 1x PBS
2. Place decapsulated testes in HEB for 30-60 minutes (on ice)
3. Remove small section of tubule and place in 20 m l of sucrose solution (100 mM)
• - dissociate tubule using tweezers
• - add additional 20 m l of sucrose solution (100 mM)
• - dissociate further using tweezers and a small pipette
• - remove large chunks of tissue
4. Dip uncoated slide into paraformaldehyde (pfm) and drain off excess liquid
• - tilt slide so drop of pfm is at one edge of the slide
• - add 20 m l sucrose/cell slurry to the drop of pfm
• - spread cells along the surface of the slide by tilting the slide
5. After adding cells immediately place slides in a humidified chamber to dry slowly
6. Remove slides from chamber after 2-3 hours
7. Wash slides 3 times (5 min each) in photoflo/ddH 2 O solution
8. Air dry slides before immunofluorescence

Hypotonic extraction buffer (HEB)

1.5 ml (30mM) Tris (pH 8.2, I M)

855.75 mg (50mM) Sucrose

250 mg (17mM) Citrate (trisodium, dihydrate)

500 m l (5mM) EDTA (500mM)

50 m l (0.5mM) DTT (500mM)

436 m l (0.1mM) PMSF (10mg/ml)

- add H 2 0 to 50 ml

- pH 8.2 – 8.4 before use

- use within 1 hr of making

100 mM Sucrose solution

0.342 g sucrose in 10 ml H 2 0

pH to 8.2

1% Paraformaldehyde

1 g Paraformaldehyde

1 drop 1N NaOH

- Dissolve pfm in 100 ml H 2 0 (at 60 ° C) , cool to room temp, pH to 9.2 with borate

- filter solution

- add 750 m l 20% triton (final conc. 0.15% triton)

0.4% Photoflo/ddH 2 O solution

800 m l photoflo in 200 mls water

pH to 8.2-8.4