Goat anti rat af647

Five-μm-thick frozen sections of kidneys were cut with Cryostat (Leica AS-LMD, Leica Biosystem,) and fixed in acetone on ice for 10 min. C3 (Hycult Biotech, HM1065), C5b-9 (Abcam, ab55811), polyclonal anti-serum to human FH (Quidel, A312), goat IgG (Jackson Immunoresearch, 005-000-003), C5aR1 (Hycult Biotech, mAb 20/70), CD31 (Abcam, ab124432), and NGAL polyclonal IgG (R&D system, AF1857) were stained for 2 h at RT and revealed by secondary Donkey anti-Goat AF647 (ThermoScientific, A-21447) or goat anti-rat AF647 (ThermoScientific, A-21434). Nucleus were stained with DAPI. Stained tissues were scanned by Slide Scanner Axio Scan™ (Zeiss) and analyzed with Zen lite™ software (Zeiss).

After fixation and washes in PBS/Triton-X (PBT) 0.5%, brains were blocked overnight in PBS/Triton-X (PBT) 0.5% supplemented with Bovine Serum Albumin (BSA) 1% and then incubated with the following antibodies: α-Imp (rabbit, 1:1000; Medioni et al., 2014 (link)), α-Imp (rat, 1:1000; Medioni et al., 2014 (link)), α-Me31B (rabbit, 1:3000; Lee et al., 2017 (link)), α-Me31B (mouse, 1:3000; Nakamura et al., 2001 (link)) α-pCamkII (rabbit, 1:1000; Santa Cruz Biotechnology, sc-12886-R), α-GFP (chicken, 1:1000; Abcam, #ab13970). After incubation with primary antibodies, brains were washed three times with PBT 0.5% and incubated overnight with secondary antibodies. The following secondary antibodies were used in this study: Goat anti-rat AF568 (Thermofisher, A-11077), Goat anti-rat AF647 (Thermofisher, A-21247) Donkey anti-rabbit AF568 (Thermofisher, A-10042), Donkey anti-rabbit AF647 (Thermofisher, A-31573), Donkey anti-mouse AF488 (Thermofisher, A-21202), Donkey anti-mouse AF568 (Thermofisher, A-10037), Donkey anti-mouse AF647 (Thermofisher, A-31571), and Goat-anti-chicken AF488 (Thermofisher, A-11039). DAPI was used at 5 μg/mL and incubated for 5 min after secondary antibody incubation. Brains were washed in PBT 0.5% three times following secondary antibody incubation and were then mounted in vectashield (Vector Laboratories).