488 conjugated donkey anti goat igg

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).

Animals were transcardially perfused with PBS and fixed with 10% formalin. The collected brains were sectioned (30-μm thick) for immunohistochemical staining. The sections were incubated overnight with the following primary antibodies: mouse anti-CD31 (1:1000, AF3628, R&D Systems, Minneapolis, MN, USA) for endothelial cells (ECs), rabbit anti-Iba-1 (1:1000, 019-19741, Wako, Richmond, VA, USA) for microglia, and goat anti-NLRP3 (1:200, MAB7578, R&D Systems) for inflammasome markers. After incubation with primary antibodies, tissues were incubated with 488-conjugated donkey anti-goat IgG (1:500, 705-545-147, Jackson ImmunoResearch, West Grove, PA, USA) for CD31, 647-conjugated donkey anti-rabbit IgG (1:500, 711-605-152, Jackson ImmunoResearch) for Iba-1, or Cy3-congugated donkey anti-rat IgG (712-165-150, Jackson ImmunoResearch) for NLRP3. Stained tissues were observed using fluorescence microscopy (Leica DM4000 B LED, LAS V4.12, Wetzlar, Germany). To quantify the intensity of CD31 and Iba-1, between two and four contralateral or ipsilateral images were acquired for each mouse under 40× fluorescence microscopy and the intensity was measured using LAS V4.12 software (Leica).