Laser scanning confocal microscopy system lsm 710

Whole-mount immunofluorescence assay was performed as described previously [25 (link)]. Briefly, larvae samples from different developmental stages of C. gigas were washed in 0.01 M PBS 3 times for 15 min each. The shells of larvae (D-shaped larvae and later) were decalcified with 5% EDTA solution in PBS for 30 min at room temperature, followed by washing 3 times for 15 min each with PBS. Afterwards, the specimens were blocked at 4°C overnight in blocking solution (10% normal goat serum, 0.25% bovine serum albumin, 1% Triton X-100, and 0.03% sodium azide in PBS) and then incubated with the rabbit anti-CgGPR1 antibody (1:50 dilution in blocking solution) at 4°C for 3 days. Subsequently, these specimens were washed 3 times for 20 min each with PBST (PBS buffer containing 0.05% Tween-20) and incubated with the goat anti-rabbit IgG antibody conjugated to Alexa Fluor 488 (1:600 dilution in blocking solution; Invitrogen) for 1 day at room temperature. The specimens were then washed 5 times for 15 min each with PBST and mounted in 80% glycerol in PBS for subsequent detection. The negative control was established by incubating the specimens with preimmune sera plus secondary antibody. Finally, all of the specimens were examined and analyzed as whole-mount using the Zeiss Laser-Scanning Confocal Microscopy System LSM 710 (Zeiss, Jena, Germany).

The whole-mount immunofluorescence assay was conducted as previously described (Thisse and Thisse, 2008 (link); Liu et al., 2015 (link)) with minor modification. In brief, the larvae stored in pure methanol were rehydrated by PBS and washed three times with PBST (PBS with 0.1% Tween20). The shells of D-shape larvae were decalcified in 25 mmol^–1 EDTA in PBS for 30 min, rinsed in PBST for 3 × 15 min, and blocked overnight in the blocking buffer (10% normal goat serum, 0.25% fetal bovine serum albumin, 1% TritonX-100 and 0.03% sodium azide in PBS). Then, the larvae were incubated with the primary antibodies (1:500 dilutions in blocking buffer) at 4°C for 3–4 days. These specimens were then washed for 3 × 15 min in PBST and incubated with goat anti-rat IgG conjugated to Alexa Fluor 488 (1:2,000 dilutions in blocking buffer; Invitrogen, Carlsbad, CA, United States) for 1 day at room temperature. After incubation in secondary antibodies, all the specimens were washed in PBST for 3 × 20 min and then mounted in 50% glycerol in PBS. The specimens incubated with pre-immune serum added with secondary antibodies were selected as negative control (Liu et al., 2015 (link)). All specimens were examined as whole-mount using the Zeiss Laser-Scanning Confocal Microscopy System LSM 710 (Zeiss, Jena, Germany).