Rabbit anti iba1 pab

Brain cryo-sections were incubated with one primary antibody followed by incubation with secondary antibody conjugated with either Alex488 or Alex555. For double-immunofluorescent staining, the same sections were then incubated with another primary antibody, followed by incubation with the appropriate secondary antibody and with Hoechst 33342 to show the nucleus. Sections were imaged using either a microscope (BX51, Olympus) equipped with a cooled CCD (DP72, Olympus) or a laser confocal microscope (Nikon A1, Japan). Data were obtained and processed using ImageJ (NIH, USA). In some cases, immunosignals were visualized by using 3,3-diaminobenzidine (Sigma-Aldrich). The following primary antibodies were used: mouse anti-GFAP mAb (1:500, Sigma-Aldrich, G3893); rabbit anti-GFAP pAb (1: 1,000; DAKO, Z0334); rabbit anti-Iba1 pAb (1:500; WAKO, 019-19741); rabbit anti-TH pAb (1:800; Chemicon, AB152); rabbit anti-GFP pAb (1:1000; Invitrogen, A11122); rabbit anti-TNFR1 pAb (1:200; Proteintech, 21574-1-AP); rabbit anti-TNFR2 pAb (1:200; Beyotime, AF8199); mouse anti-RGS5 pAb (1:500; Sigma-Aldrich, sc-390245).

The IHC or IF staining was as described previously^{25 (link),26 (link)}. Experimental mice were anesthetized and perfused transcardially with PBS followed by 4% paraformaldehyde. Brain samples were postfixed with 4% paraformaldehyde overnight and equilibrated in 30% sucrose. Coronal sections of 10 μm were prepared with a sliding microtome, and were then incubated with primary antibodies: rabbit anti-tyrosine hydroxylase pAb (1:500; Chemicon); rabbit anti-Iba1 pAb (1:500; WAKO); rabbit anti-GFAP pAb (1:800; DAKO); mouse anti-GFAP mAb (1:1,000, Sigma-Aldrich). The brain slices were then incubated with the horseradish peroxidase (HRP)- or fluorescence-conjugated secondary antibodies. The peroxidase activity of immune complexes was revealed with a DAB kit according to the manufacturer’s instruction (Beyotime, P0203). Sections were imaged using either a cooled CCD (DP72, Olympus) on a microscope (BX51; Olympus).