Alexa fluor 555 goat anti mouse igg2a

BmN4 cells were placed on 0.075% poly-L-lysine coated glass coverslips. The cells were treated with or without 35 μg/mL digitonin in PBS for 5 min, fixed with 4% formaldehyde in PBS for 15 min, and permeabilized with 0.1% Triton X-100 in PBS for 15 min. After blocking with 3% bovine serum albumin (BSA) in PBS, the cells were incubated with primary antibodies diluted with 3% BSA in PBS at room temperature. Anti-Mael (this study), anti-Siwi, anti-Spn-E, anti-Qin, anti-Vasa (Nishida et al., 2015 (link)), anti-Vret (Nishida et al., 2020 (link)), anti-Flag (Sigma-Aldrich), and anti-Myc (Sigma-Aldrich) antibodies were used as primary antibodies. After washing with PBS, the cells were incubated with secondary antibodies diluted in PBS with 3% BSA at room temperature in the dark. Alexa Fluor 488 goat anti-mouse IgG1, Alexa Fluor 555 goat anti-mouse IgG1, Alexa Fluor 555 goat anti-mouse IgG2a, and Alexa Fluor 488 goat anti-rabbit IgG antibodies (Thermo Fisher Scientific) were used as secondary antibodies. After washing with PBS, the cells were mounted with VECTASHIELD Antifade Mounting Medium with DAPI (VECTOR LABORATORIES). Images were collected using a Zeiss LSM980 laser-scanning microscope with a Plan-Apochromat 63×/1.4 Oil DIC objective lens. Pixel dwell time was set at 2.05 μs and two sequentially scanned images were averaged.