Alexa fluor 488 conjugated goat anti rat antibody

Anesthetized mice were perfused with 20 mL of PBS. Eyes were enucleated and fixed in 4% paraformaldehyde in 2× PBS for 15 min and then transferred to 2× PBS on ice for 10 min. After dissecting eyes, retinal whole mounts were prepared. Retinas were then transferred to ice-cold methanol and kept at −80 °C until use.
For immunohistochemistry, retinas were first blocked in a blocking buffer (0.3% Triton, 0.2% BSA, and 5% goat serum in PBS) for 1 h at room temperature and incubated with primary antibodies and Alexa Fluor 647-conjugated isolectin GS-B4 (1:100; Thermo Fisher Scientific) overnight at 4 °C. After washing, retinas were incubated with secondary antibodies for 4 h at 4 °C. Retinas were mounted after washing. Rabbit anti-P2ry12 antibody (1:500; a gift from H. Weiner, Brigham and Women’s Hospital), rat anti-CD11b antibody (1:100, clone M1/70; Abcam), rat anti-MHC class II (1:1,000, I-A/I-E; BD Pharmingen), rat anti-CD4 (1:200, RM4-5; BD Pharmingen), and rat anti-CD8a (1:200, 53-6.7; BD Pharmingen) were used for primary antibodies. Alexa Fluor 594-conjugated goat anti-rabbit antibody, and Alexa Fluor 488-conjugated goat anti-rat antibody (1:500; Thermo Fisher Scientific) were used for secondary antibodies.

Cells were synchronized to the S phase by double thymidine block. Subsequently, CIdU (25 μM) was added for 20 min, followed by IdU (250 μM) for a further 40 min. Cells were harvested and washed twice with PBS. Cell samples of 2 μL (1,000 cells) were spotted onto the glass slide, air-dried for 5 min, and lysed in 7 μL lysis buffer (200 mM Tris-HCl, pH 7.5, 50 mM EDTA, 5% SDS) for 2 min. The slide was placed at an angle of 30 °C to allow the DNA to flow out slowly. Slides were air dried before fixation in methanol: acetic acid (3:1). Fixed fibers were denatured in 2.5 M HCl for 1 h before blocking in 5% BSA for 1 h and then incubated with anti-mouse BrdU antibody (BD Biosciences, 1:60), and anti-rabbit BrdU antibody (Sigma, 1:500) for 2 h, respectively. Slides were washed three times with 0.05% Tween-20 and PBS, followed by incubation with Alexa Fluor 488-conjugated goat anti-rat antibody (Thermo Fisher Scientific, 1:400) and Alexa Fluor 647-conjugated goat anti-mouse secondary antibody (Thermo Fisher Scientific, 1:400) for 1 h in the dark. Slides were washed three times with 0.05% Tween-20 and PBS. Slides were imaged using an Olympus microscope and the track lengths of the labeled replication forks were analyzed manually using Image J software.