Visualizing Germline Stress Granules

To visualize germline SGs, immunostaining against CGH-1 and GFP was performed as previously reported by (Huelgas-Morales et al., 2016 (link)) with some modifications. The gonads of one-day-old animals were dissected, freeze cracked, fixed in cold methanol for 1 min, and then treated with 3.3% paraformaldehyde for 15 min. The samples were washed twice with PBT, and were then blocked in PBT containing 30% of normal goat serum (NGS; Sigma-Aldrich, St. Louis, MO) for 30 min. Primary antibody incubation was performed overnight at 4°C with rabbit anti-CGH-1 (1:1000) (Boag et al., 2005 (link)) and mouse anti-GFP (1:50) (A11120 from Molecular Probes, Eugene, OR). Secondary antibody incubations were performed for 2 h at room temperature with Alexa Fluor 594-conjugated anti-rabbit IgG and Alexa Fluor 488-conjugated anti-mouse IgG (1:100; H + L; A11001, Molecular Probes, Eugene, OR). To detect DNA, 1 ng/ul 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI) was used. Vectashield (Vector laboratories, Burlingame, CA) was added to avoid photo bleaching before sealing the sample.