Rabbit anti iba1

After washed with PBS (5 min each). Prepared brain sections were permeated with 0.5% Triton X-100 for 30 min, and blocked with 10% goat serum for 2 h at room temperature, followed by overnight incubation with primary antibody at 4°C. The next day, samples were incubated with the corresponding secondary antibody at room temperature for 2 h. And 4′, 6-diamino-2-phenylindole (DAPI; Boster, Wuhan, China) were counterstained for 5 min. Finally, stained sections were examined, and images were captured using a confocal microscope (LSM-880; Zeiss). The primary antibodies used in the experiment are as follows: goat anti-ionized calcium-binding adaptor molecule (Iba1; 1:200; Abcam, UK), rabbit anti-Iba1 (1:200; GeneTex, Irvine, CA, USA), mouse anti-CD86 (1:200; Santa Cruz, CA, USA), rabbit anti-CD206 (1:200; Abcam, UK). Images from four sections of the brain around the PAG, VTA and VPL were captured using a 20× objective on a Zeiss confocal microscope (Zeiss, LSM780, Germany). Cell numbers were calculated per random microscopic field (100 × magnification). All counts were performed in a blinded fashion.

As reported earlier [38 (link)], TI-induced immunoreactive pyramidal neurons and activated glial cells in the CA1 domain of the hippocampus were evaluated using immunohistochemistry (IHC). Briefly, to block endogenous peroxidase, cryo-sections of the brain were dipped into a 0.3% hydrogen peroxide (H₂O₂) solution. The brain sections were then blocked using 5% normal goat serum for GFAP, 4HNE and Iba-1 or horse serum for NeuN. Blocks of brain tissue were incubated with a variety of antibodies overnight at 4 °C, together with rabbit anti-GFAP (1:1000, Gene tex, Irvine, CA, USA), mouse anti-NeuN (1:800, Chemicon International, Temecula, CA, USA), rabbit anti-Iba-1 (1:1000, Gene tex, CA, USA), and rabbit anti-4HNE (1:800, Abcam, Cambridge, UK), to identify activated astrocytes, mature neurons, and microglia, respectively. The brain sections were then treated with secondary antibodies (Vector Laboratories Inc., Newark, CA, USA), Vectastain ABC (Vector Laboratories Inc.), and diaminobenzidine chromogen, and coverslips were glued with Canada balsam (Kanto Chemical, Tokyo, Japan).