OPP-click

OPP Click-iT in C. elegans germlines

Reagents

• Click-it Plus OPP Alexa Fluor 488 Protein Synthesis Assay Kit (Invitrogen Cat# C10456).

• Emetine (Sigma Cat# 324693)

• Anisomycin (Sigma Cat# A9789)

Solutions

• Egg buffer [25 mM HEPES pH 7.3, 118 mM NaCl, 48 mM KCl, 2 mM CaCl₂, 2 mM MgCl₂]

• Anisomycin stock solution (1,000x)
10 mg/ml (or 37 mM) in 100% ethanol solution
Store at -20 °C

• Emetine dihydrochloride stock solution (1,000x)
25 mg/ml (or 45 mM) in 50% ethanol
Store at -20 °C

Anisomycin and emetine concentrations recommended by Bastide et al., 2018

1. Allow Click-it kit reagents to thaw to room temperature before beginning.

2. Transfer adult worms into a glass dish containing egg buffer with 1mM levamisole.

3. Dissect out the germlines by cutting off the heads with two 25-gauge needles. The germlines should pop out of the worm. Finish dissecting within 5 minutes so the germlines aren’t sitting around for too long.

4. Once the germlines are dissected, remove as much buffer as possible and resuspend them in egg buffer alone, or with 45 µm emetine, or 37 µm anisomycin. Incubate 15 minutes.

5. Remove as much of the previous buffer as possible. Add egg buffer containing 20 µM O-propargyl-puromycin (OPP) (Plus the same concentration of emetine or anisomycin as before) and incubate for 5 minutes.

6. Rinse germlines once in PBS and then fix in 4% formaldehyde diluted in PBS for 15 minutes.

7. Transfer germlines to an Eppendorf tube with a glass Pasteur pipet. In between each wash step, spin germlines down at 3,000 RPM for 1 minute or let them settle to the bottom of the tube by gravity.

8. Remove fix and add 0.5% Triton® X-100 in PBS and incubate for 15 minutes at room temperature.

9. Prepare 1X Click-iT® OPP Reaction Buffer Additive by diluting the 10X solution 1:10 in deionized water. Prepare this solution fresh and use the solution on the same day.

10. Prepare Click-iT® Plus OPP reaction cocktail:

11. Remove the permeabilization buffer (from step 8) and wash cells twice with PBS. Remove the wash solution.

12. Add 200 μL per tube of Click-iT® Plus OPP reaction cocktail to each well and mix well.

13. Incubate for 30 minutes at room temperature, protected from light.

14. Remove the reaction cocktail and wash with Click-iT® Reaction Rinse Buffer (Component F). Remove the rinse buffer.

15. Wash again in PBS. Remove as much PBS as possible.

16. Resuspend germlines in a drop of ProLong™ Glass Antifade Mountant with NucBlue™ Stain mounting media. Pipet onto glass slides. Gently place coverslip on top. Let cure overnight.

References
Bastide A, Yewdell JW, David A. 2018. The RiboPuromycylation method (RPM): an immunofluorescence technique to map translation sites at the Sub-cellular level. Bio-Protocol 8:e2669.