Alexa fluor 555 donkey anti mouse secondary antibody

Immunofluorescence staining was performed as previously described (19 (link)). Briefly, cell monolayers were fixed with 4% paraformaldehyde for 10 min and permeabilized with 0.01% Triton X-100 in PBS at room temperature for 10 min. After blocking with 3% BSA at room temperature for 1 h, cells were incubated with a primary anti-RANKL mouse monoclonal antibody (1:50; cat. no. ab45039; Abcam) at 4°C overnight, followed by incubation with an Alexa Fluor 555 donkey anti-mouse secondary antibody (1:500; cat. no. ab150106; Abcam) in the dark at room temperature for 1 h. The coverslips were mounted using DAPI solution (Invitrogen; Thermo Fisher Scientific, Inc.). Slides were viewed using a fluorescent microscope (Imager Z1; Zeiss AG). The fluorescence intensity was analyzed using Image-Pro Plus 6.0 (Media Cybernetics, Inc.).

Paraffin-embedded sections of immature COCs were processed as previously described (31 (link)). A group of 50–60 immature COCs was collected, fixed with 4% paraformaldehyde (Solarbio, P1110) at 4°C for 12 h, and subsequently processed. After fixation, the COCs were dehydrated in 70%, 95%, and 100% alcohol at room temperature for 30 min, followed by incubation in xylene for 30 min. The COCs were then embedded in soft wax and hard wax, respectively. These paraffin blocks were cut into 5-µm thick sections, blocked with 5% normal donkey serum (Jackson ImmunoResearch Laboratories, 017-000-121), and incubated with anti-CX43 (1:100, Thermo Fisher, monoclonal, 13–8300) for 1 h at 37°C, followed by detection using Alexa Fluor® 555 donkey anti-mouse secondary antibody (1:800, Abcam, 150110) for 1 h at 37°C. The sections were counterstained with 4′,6-diamidino-2-phenylindole (DAPI, 2 µg/mL, Abcam, ab104139). Images were captured using the Nikon C2 confocal microscope (Nikon, Tokyo, Japan) and analyzed using NIS-Elements software. Antibody specificity was verified (Supplementary Fig. 1, see section on supplementary materials given at the end of this article).