Cy3 conjugated rabbit anti rat igg

Microscopy was carried out in Olympus FluoView FV1000 confocal microscope. Alexa Fluor 488 labeled phalloidin (Invitrogen, Carlsbad, CA, USA) was used to visualize F-actin. CR3 was visualized using two rat anti-mouse CR3 mAbs produced by hybridoma cell lines: mAb M1/70 (Developmental Studies Hybridoma Bank, Iowa City, IA, USA) and mAb 5C6 (American Type Culture Collection, Rockville, VA, USA) followed by Cy3-conjugated rabbit anti-rat IgG (Jackson ImmunoResearch, West Grove, PA, USA). Optical slices, 1 μm thick, were scanned sequentially and then used to reconstruct whole images.

Immunofluorescent staining was performed as previously described [25 (link)]. Briefly, tissue sections or cells were blocked using a 10% bovine serum albumin solution with 0.25% triton X-100 for 1 h at room temperature, followed by incubation overnight at 4 °C with primary antibodies. These primary antibodies included rabbit anti-NeuN (1:500, Millipore), rabbit anti-GFAP (1:500, Cell Signaling Technology), rabbit anti-IBA-1 (1:500, Wako Pure Chemical Industries), goat anti-IBA-1 (1:500, Wako Pure Chemical Industries), mouse anti-GSDMD (1:50, Santa-Cruz), mouse anti-ASC (1:200, Santa-Cruz), mouse anti-caspase-1 (1:200, Santa-Cruz), mouse anti-NLRP3 (1:200, AdipoGen), rabbit anti-Hv1 (1:50, Sigma), and mouse anti-8-OHdG (1:200, Abcam). Next, samples were washed in PBS and incubated at room temperature for 1 h with the appropriate secondary antibodies, as follows: FITC-conjugated goat anti-mouse immunoglobulin G (IgG), Cy3-conjugated goat anti-rabbit IgG, Cy3-conjugated rabbit anti goat IgG, Cy3-conjugated rabbit anti-rat IgG, and 488-conjugated donkey anti-goat IgG (Jackson ImmunoResearch, West Grove, PA, USA). Finally, sections were stained with 4,6-diamidino-2-phenylindole (DAPI). Samples were imaged using a confocal microscope (Olympus, BX51).