1. Patch cells (-80C) onto a YPG plate to eliminate petites, O./N.. Streak cells onto a YPD plate for about 2 days.

2. Follow the protocol for obtaining synchronous G0/G1 cells in SK1.

3. Collect cells enriched at pachytene (~5 hours for wt at 30C) (10ml).

4. Wash once with 1ml 1% KOAC +1M sorbital, pH 7.0.

5. Spheroplast cells in 1ml buffer (same as above): add 10ml 1M DTT, wait for 10 minutes, then add 10ml 10mg/ml Oxylyticase, incubate at 30C (23C and 34C for ts mutants) for 20-30 minutes. Monitor spheroplast with phase microscopy (10% Sarcosyl should burst the cells).

6. Spin down with a desktop centrifuge for 1 minute.

7. Wash once in ice cold 1X MES +1M sorbitol (MES buffer: 0.1M MES (2-(N-morpholino) ethane sulfonic acid); 1mM EDTA; 0.5mM MgCl2 (pH 6.4)).

8. Suspend in 200 ml 1X MES buffer.

9. Add 720ml fixation buffer: 720ml 1X MES +4% Para-formaldehyde (final concentration. Alternatively, add 720ul PHEM + 4% PFA (PHEM buffer: 60 mM Pipes, 25 mM Hepes, pH 6.95. 10 mM EGTA, 4 mM MgCl2). Pour onto slides (Sigma, Gold Seal, Cat No. 3063, not necessary to be acid-washed), wait for 20-30 minutes (cover slide with a 22X60mm coverslip).

10. Rinse with 5 ml 0.4% (vol/vol) PhotoFlo 200 (Kodak), air dry.

11. Wash once in PBS

12. Block slides with 3% BSA + 5% Goat serum in PBS with 0.05% NaN3 (from 2 hours to O./N. at room temperature).

13. Add primary antibody: anti-HA (12CA5) 1:100-200, anti-Zip1: 1:100, etc. in 3%BSA, PBS with 0.05% NaN3. Incubate at room temperature for at least 2 hours.

14. Wash at least 3X in PBS.

15. Appropriate secondary antibodies(usually 1:100 dilution) in 3%BSA + PBS + 0.05% NaN3.

16. Wash 3X in PBS.

17. Add mounting medium (with Dapi) to the slides. Store at -20C.

Reference: Dresser, ME and CN, Giroux, 1988, JCB, 10b:567-573
Engebrecht, J and SR, Roeder, 1990, MCB, 10, 2379-2389