（Source：NonetLab）
1. Wash worms off with 1X PBS
2. Spin Down Worms in 12ml glass conical tubes. Wash 3-4 times with PBS until all E.coli are gone (clear). Make sure that you resuspend all worms thoroughly each time.
3. Aspirate as much of the 1x PBS as possible
4. Add a mix of 400ul Bouin's +400ul MeOH + 10ul BME. Alternately 4% paraformadehyde +400ul MeOH + 10ul BME depending on the Ab. For a small plate of worms 200+200+5 is enough.
5. Transfer worms to a 1.5ml centrifuge tube w/ a glass pipette. For sensitive Abs precool eppendrof tube, fix and tube of worms.
6. Rock tubes 15 min in rotary shaker at room temp or 4C depending on Ab. For sensitive Abs do only 7-10 min.
7. Quick freeze in liquid N2 and keep at -80 C for minimum 2 hours (can be kept for months with little problem). For problem Abs I have had best results with O/N at -80C
8. Quick Thaw under running hot H2O until it melts (but before it is room temp.)
9. Rock tubes 15 min (10 min for problem Ab).
10. Do 4-5 BT washes. 1st BT wash is a rinse and all subsequent ones are 3-5 min. washes. Typically I do spin at 1-2 K for 30 seconds.
11. Make BTB: 1x Borate Buffer, 0.5% Triton X-100, 2% BME. Avoid using if over 2 weeks old.
12. Spin, aspirate BT
13. Add BTB, rock a few seconds to mix
14. Repeat steps 11-12 once more
15. Resuspend worms in BTB and incubate for 1 hr with rotation/rocking (best is 30 min x2 and then every hour for 2-4 hours.)
16. Wash again w/ BTB and incubate 2-3hrs. If permeablization is a problem do a harsh spin i.e. at 14k for 10-15 seconds coming down slowly to 1-2 K.
17. Quick rinse with AbA or PBST
18. Block for 30 min- 1 hr. with PBST/AbA. Remove as much of soln and add Primary Ab
19. Incubate in 4C O/N, no shaking needed. Some Abs only work if you do 3 o/n at 4C and on the last day a few hours at room temp. before beginning washes.
20. Wash 5x for 5-10 min in AbA. For better staining do 6-8 changes in 3-6 hours.
21. Remove most of the AbA