Alexa fluor 594 donkey anti rabbit

Cells were plated on clean cover slides in 100-mm culture dishes. When the cells reached 70–80% confluence, the cells on the slides were washed twice with ice-cold 1× PBS and fixed with 4% paraformaldehyde for 10 min at room temperature. The cells were washed three times with 1× PBS at room temperature, after which the cover slide was transferred to a humid dish. Next, 1% BSA was utilized to block the cells at room temperature for 30 min. The cells were washed cells three times with 1× PBS and incubated overnight at 4 °C with 150 μL of primary antibodies diluted in PBST. The primary antibodies were anti-Ki67 (1:400, Abcam, ab15580), anti-γH2AX (1:300, CST, #9718P), and anti-53BP1 (1:200, Abcam, ab36823). The cells were washed 4 times with PBST and incubated at room temperature for 2 h with 150 μL of secondary antibodies (1:500, Alexa Fluor 488 Donkey Anti-Mouse or Alexa Fluor 594 Donkey Anti-Rabbit, Life Technologies) diluted in PBST. The cells were washed cells three times with PBST and incubated with 1 ng/μL of DAPI at room temperature for 3 min. The cells were washed 3 times with 1× PBS, followed by a wash with ddH₂O, after which the cover slide was sealed with 10 μL of Fluoromount-G. After 1 h, images of the cells were captured under a fluorescent microscope. The immunofluorescent signals were examined using Image J software (NIH).

Mice brains were dissected at P1 and then processed as described in the iDISCO protocol^{20 (link)}. Briefly, after tissue collection and fixation, a pretreatment with methanol was performed before immunostaining. Then, permeabilization and blocking of the tissue were performed before incubation in primary antibodies at the following concentration: rabbit anti-TH (Pel-Freez, 1:250) for 7 days. After 1 day of washing in buffer PTwH (100 ml PBS 10×, 2 ml Tween-20, 1 ml of 10 mg/ml heparin stock solution), brains were incubated in the secondary antibody Alexa Fluor 594 donkey anti-rabbit (Life Technologies, 1:400) for 6 days. After washing, brains were transferred in a clearing solution for further imaging.

Anti-LC3A/B rabbit polyclonal antibody was purchased from Cell Signaling Technology. Anti-p62 mouse monoclonal antibody and Alexa®Fluor 647-labeled Anti-LAMP1 mouse monoclonal antibody were purchased from Santa Cruz Biotechnology. Anti-ATPB mouse monoclonal antibody was purchased from ABCAM. Anti-LAMP3 (CD63) mouse monoclonal antibody was purchased from BD Pharmingen. Alexa®Fluor 594 donkey-anti-rabbit, Alexa®Fluor 594 goat-anti-mouse secondary antibodies, anti-GFP rabbit monoclonal antibody, and goat-anti-mouse secondary antibody HRP were purchased from Life Technologies. Anti-ATG5 mouse monoclonal antibody was purchased from MBL. Anti-GAPDH mouse monoclonal antibody was purchased from Sigma Aldrich. Pooled Ig from HIV-1-infected patients (HIV-Ig) was obtained from the NIH AIDS Research and Reference Reagent Program. Rapamycin and leupeptin were purchased from Sigma Aldrich. Bafilomycin A1 was purchased from Cell Signaling Technology. DAPI was purchased from Life Technologies.