Immunostaining

2.2.1. Whole Mount Immunohistochemistry

1. Testis buffer or TB1 (see item 1, Subheading 2.1.). For monoclonal antibodies directed against phosphorylated epitopes, use TBPi buffer: 10 mM Tris-HCl, pH 6.8, 180 mM KCl, 50 mM NaF, 1 mM Na3VO4, 10 mM Na P-glycerophosphate.

2. 4% Formaldehyde (from 40% stock) in phosphate-buffered saline (PBS), or make up fresh 4% paraformaldehyde in PBS or HEPES buffer: 100 mM HEPES, pH 6.9, 2 mM MgSO4, 1 mM EGTA. HEPES buffer may be stored at -20°C, but check pH before (re)use.

4. Fetal calf serum (FCS). Sterile, but out of date will do; it is used as a blocking agent.

5. 24-Well tissue culture plates and tissue culture inserts with 8-|im mesh (Falcon no. 3097).

6. Primary antibody.

7. Secondary antibody, biotin-conjugated (Vector Laboratories).

8. ExtrAvidin-HRP conjugate (Sigma-Aldrich), or Vectastain ABC reagents (Vector Laboratories).

9. 3,3'-Diaminobenzidine (DAB; available in tablet form from Sigma). DAB is a potent carcinogen. Wear gloves and use caution.

10. Hydrogen peroxide.

11. Microscope slides and cover slips.

12. Mounting medium: 85% Glycerol. Add 2.5% «-propyl-gallate if counterstaining with Hoechst.

13. Hoechst 33258 (e.g., Sigma-Aldrich) (optional; see Subheading 3.2.1.).

2.2.2. Formaldehyde Fixation for Confocal Microscopy

1. Testis buffer or TB1 (see item 1, Subheading 2.1.).

2. Liquid nitrogen, scalpel, safety glasses.

3. 100% Ethanol (chilled on dry ice).

4. 4% Formaldehyde in PBS.

6. PBST-DOC: PBS + 0.3% Triton X-100 + 0.3% sodium deoxycholate (from 10% stock).

7. FCS (sterile, but does not have to be in date), or bovine serum albumin (BSA).

8. Poly-L-lysine-coated slides.

9. Siliconized 22 x 22-mm2 cover slips.

10. Evostick impact adhesive or rubber cement.

11. Clear nail polish.

12. Humid chamber (e.g., sandwich box with wet tissue paper).

13. Primary antibody.

14. Fluorescently conjugated secondary antibody (Jackson ImmunoResearch Laboratories or Vector Laboratories).

15. Propidium iodide or DAPI (4,6-diamidino-2-phenylindole) if staining for DNA.

16. RNAse A, 10 mg/mL stock (store at -20°C). Required if using propidium iodide.

2.2.3. Methanol/Acetone Fixation for Confocal Microscopy

As per Subheading 2.2.2., except replace item 3 (ethanol) with methanol cooled on dry ice, item 4 (4% formaldehyde) with acetone cooled on dry ice, and items 5 and 6 (PBST and PBS-DOC) with PBS + 1% Triton X-100 + 0.5% acetic acid (PBS-T-AA).

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