Alexa fluor 488 donkey anti rabbit secondary antibody

Cells were planted on the glass coverslips a day in advance. After transfected with indicated plasmids, the cells were fixed with 4% paraformaldehyde. The cell membranes were p permeabilized with NP40, then blocked with blocking buffer for 1 h. After incubation with primary antibody against Myc-tag (Proteintech, 16286-1-AP) for overnight, cells were washed with Phosphate-Buffered Saline (PBS)-Tween and incubated with Alexa Fluor 488 donkey anti-rabbit secondary antibody (Invitrogen, R37118). Mitochondrial were stained with MitoTracker (Invitrogen, M22426) for 30 min and nuclei were stained with DAPI (Beyotime, C1002) for 10 min. The slides were observed using the Lecia Tcs SP8 confocal microscope.

Rabbit anti-FPR2 antibody (Novus), mouse anti-CD68 (Abcam) and mouse anti-MPO (Abcam) were used as primary antibodies and were separately detected by Alexa Fluor 488 donkey anti-rabbit secondary antibody (1:1000; Invitrogen) and Alexa Fluor 594 donkey anti-mouse antibody (1:200, Invitrogen). For nuclear staining, 4′,6-diamidino-2-phenylindole (DAPI) was used.

Mouse cochleae were embedded in OCT compound and sectioned in 8–10 μm thickness. After fixation with 4% PFA in PBS for 30 min, samples were permeabilized and blocked with PBT1 for 30 min, then incubated with rabbit anti-CDK5 antibody (Santa Cruz, Cat. No. sc-173, 1:50 diluted) in PBT1 overnight at 4°C. Afterward, samples were incubated with Alexa Fluor^® 488 donkey anti-rabbit secondary antibody (Invitrogen, Cat. No. A21206, 1:200 diluted) in PBT2 for 1 h, followed by incubation with DAPI (Gene View Scientific Inc.) in PBS for 1 h. Lastly, samples were mounted in PBS/glycerol (1:1) and imaged with a confocal microscope (LSM 700, Zeiss, Germany).

All steps were performed at room temperature unless otherwise indicated. Dissected organ of Corti explants were fixed with 4% paraformaldehyde (PFA) in PBS for 30 min, followed by permeabilization and blocking with PBT1 (0.1% Triton X-100, 1% BSA, and 5% heat-inactivated goat serum in PBS, pH 7.3) for 30 min. Samples were then incubated with rabbit anti-CDK5 antibody (Santa Cruz, Cat. No. sc-173, 1:100 diluted) or rabbit anti-MYO6 (Cell Signaling Technology, Cat. No. 9200, 1:50 diluted) in PBT1 overnight at 4°C, followed by incubation with Alexa Fluor^® 488 donkey anti-rabbit secondary antibody (Invitrogen, Cat. No. A21206, 1:200 diluted) in PBT2 (0.1% Triton X-100 and 0.1% BSA in PBS) for 1 h. After that, samples were incubated with TRITC-conjugated phalloidin (Sigma-Aldrich, Cat. No. P1951) in PBS for 30 min, then mounted in PBS/glycerol (1:1) and imaged with a confocal microscope (LSM 700, Zeiss, Germany). For CtBP2 staining, samples were incubated with mouse anti-CtBP2 antibody (BD, Cat. No. 612044, 1:100 diluted) in PBT1 overnight at 4°C, followed by incubation with Alexa Fluor^® 568 goat anti-mouse IgG1 (Invitrogen, Cat. No. A21124, 1:200 diluted) in PBT2 for 1 h and DAPI (Gene View Scientific Inc.) in PBS for 1 h.

Bone marrow neutrophils were plated on coverslips in a 24-well plate for 2 h, followed by stimulation with 10% pre-I/R lymph, 10% post-I/R lymph, or media for 1 h. After fixation, permeabilization, and blocking, the cells were stained with p-p65 or p65 antibody overnight at 4°C, followed by incubation with an Alexa Fluor 488 donkey anti-rabbit secondary antibody (Invitrogen, A21206) for 1 h at room temperature. The nuclei were stained with DAPI, and images were taken using a Leica SP8 confocal microscope.

8-μm sagittal sections prepared from the formalin-fixed paraffin-embedded cerebellum were deparaffinized in xylene and rehydrated in gradient ethanol. Antigen retrieval was performed by microwave heating in 10 mM sodium citrate buffer. Non-specific binding sites were blocked by 5% BSA for 1 h at room temperature. The sections were incubated with primary antibody (1:500) against SCT (Abmart, Shanghai, China), SCTR (Abmart, Shanghai, China), or Calbindin D-28K (Abcam, Cambridge, UK) overnight at 4°C and then Alexa Fluor 488 donkey anti-rabbit secondary antibody (1:500; Invitrogen) for 1 h at room temperature. The sections were counterstained with 0.5 μg/mL Hoechst 33258 (Invitrogen). Images were captured using a Nikon 80i fluorescent microscope (Nikon, Tokyo, Japan).