Goat anti rabbit igg antibody conjugated to alexa fluor 488

Testicular tissues obtained were fixed in 4% paraformaldehyde (PFA) and embedded in paraffin. Embedded blocks were sectioned at 2 μm and were immersed in xylene I and II for 8 min, separately. Next, the samples were successively immersed in 100%, 95%, and 75% alcohol for 5 min each and finally rinsed in deionized water for 5 min. Antigen retrieval was achieved in boiling sodium citrate buffer for 10 to 15 min, and then, the samples were washed three times in 1 × PBS for 5 min each after they cooled to room temperature (RT). Then, the samples were blocked in blocking solution (1 × PBS + 1% BSA) for 30 min and incubated with primary antibody against Lin28a (1:50, Santa Cruz) overnight at 4 °C. Sections were then washed in 1 × PBS three times and incubated with a goat anti-rabbit IgG antibody conjugated to Alexa Fluor 488 (Invitrogen) for 1 h at RT. Coverslips were mounted onto slides with Anti-fading Buffer mounting medium (Bioworld) containing 1 μg/ml Hoechst33342 (Sigma)49 (link)51 (link).

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).