Rabbit anti tnfr2 pab

Western blotting was performed as described previously [30 (link)]. The following primary antibodies were used: mouse anti-β-actin mAb (1:5000; Sigma-Aldrich, A5441); rabbit anti-IL-1β pAb (1:1000; Abcam, ab9722); rabbit anti-TNF-α pAb (1:1000; Abcam, ab9739); rabbit anti-GAPDH pAb (1:5000; Proteintech, 10494-1-AP); rabbit anti-alpha-synuclein pAb (1:2000; Cell Signaling, 2628); rabbit anti-TNFR2 pAb (1:1000; Beyotime, AF8199); rabbit anti-HA tag pAb (1: 1000; Abcam, ab9110); mouse anti-FLAG tag mAb (1: 5000; Sigma-Aldrich, F1804); The membrane was washed and incubated for 2 h at room temperature with the corresponding secondary antibodies: (a) HRP-conjugated goat anti-rabbit IgG (1:10,000; Jackson ImmunoResearch Laboratories, 115-035-003); (b) HRP-conjugated goat anti-mouse IgG (1:10,000; Jackson ImmunoResearch Laboratories, 115-035-003). Peroxidase activity was detected with SuperSignal WestPico chemiluminescent substrate (Pierce Biotechnology) and visualized and digitized with ImageQuant (LAS-4000, Fujifilm, Japan). Optical densities of bands were analyzed by using ImageReader software (Fujifilm, Japan). Protein levels, quantified by computer analysis as the ratio between each immunoreactive band and the levels of β-actin or GAPDH, were expressed as a percentage of vehicle-treated control.

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).