FISH Staining of C. elegans Intestines

C. elegans intestines were dissected as described [55 ]. Briefly, adults were transferred to a drop of M9 with levamisole on a microscope slide and the heads and tails were cut with a 25-gauge needle to extract the intestines. Samples were transferred to a microfuge tube and fixed with 4% paraformaldehyde for 1–2 h. FISH was then performed essentially as described for bacteria [56 (link)]. Samples were washed with PBS + 0.1% Tween 20 and then transferred to hybridization buffer (900 mM NaCl, 20 mM Tris [pH 7.5], 0.01% SDS) containing 5 ng/μl probe. Probes were designed against regions of the ribosomal sequence specific to N. parisii and were synthesized with a Quasar 570 (Cy3) 5′ modification and HPLC purified by Biosearch Technologies, Inc. Two N. parisii-specific probes were tested and gave similar results: MicroA (CTCTGTCCATCCTCGGCAA) and MicroB (CTCTCGGCACTCCTTCCTG). These probes also cross-react with the Nematocida sp. 1 sequence isolated from JU1348, the infected C. briggsae strain from India. The universal bacterial probe EUB338 (GCTGCCTCCCGTAGGAGT) was synthesized the same way. Hybridization was performed at 46 °C overnight. Intestines were washed at 48 °C for 1 h in wash buffer (900 mM NaCl, 20 mM Tris [pH 7.5], 0.01% SDS, 5 mM EDTA) and then mounted for microscopy with Vectashield containing DAPI (Vector Laboratories). For Figure 3A–3C, staining was performed in parallel and exposure times were the same for all. FISH staining was also performed as above, except without intestine dissection and with acetone fixation for 15 min at room temperature [57 (link)] instead of paraformaldehyde fixation. These conditions allowed for better staining of spores and were used for the FISH staining shown in Figure 3E and Figure 8G–8I.