3 3 diaminobenzidine tablets d4293

Aortic root serial cryosections (5 µm thick) were cut and stained for macrophages (rat antimouse CD68, dilution 1:25; catalog no. MCA1957; Bio-Rad) or T cells (rabbit antimouse CD3, dilution 1:50; catalog no. MA1-90582; Thermo Scientific). After primary antibody incubation (overnight at 4 °C) either a secondary antirat IgG horseradish peroxidase-conjugated antibody (dilution 1:100; catalog no. STAR72; Bio-Rad) or goat antirabbit IgG horseradish peroxidase-conjugated antibody (dilution 1:100; catalog no. 1705046; Bio-Rad) were incubated for 1 h at RT. Peroxidase activity was detected using 3,3′-diaminobenzidine tablets (D4293; Sigma-Aldrich), and sections were counterstained with hematoxylin (catalog no. 3530-32; Ricca Chemical). Negative control was performed by omitting the primary antibody. Images were acquired using the BZ-X800 microscope (Keyence), and the quantification of stained area was performed with Image Pro Plus (v.9; Media Cybernetics). Results were expressed as average of three to five sections per mouse. Normal distribution of the data was confirmed using the Shapiro–Wilk test and Kolmogorov–Smirnov test. Statistical analysis was performed by one-way ANOVA with Tukey’s post hoc test.

Mice were anesthetized with isoflurane and perfused with ice-cold PBS. Brains were fixed with ice-cold 4% (w/v) paraformaldehyde overnight and cryoprotected in 30% (w/v) sucrose (in PBS) overnight at 4 °C. Thirty-five μm coronal sections were probed with the primary antibodies in the blocking buffer (PBS with 10% (v/v) goat serum and 0.1% (v/v) Triton X-100) overnight at 4 °C and then incubated with the appropriate fluorescence-conjugated secondary antibodies at 25 °C for 1 h. Images were acquired using a confocal microscope (Leica, Wetzlar, Germany).
For TH counting, sections were probed with rabbit anti-TH (NB300-109, Novus Biologicals, Centennial, CO, USA) in the blocking buffer overnight at 4 °C and then incubated with biotin-SP-AffiniPure goat anti-rabbit IgG H+L (111-065-045, Jackson ImmunoResearch, Philadelphia, PA, USA) at 25 °C for 1 h. We used streptavidin-conjugated HRP (Vectastain ABC kit, PK4000, Vector Laboratories, Burlingame, CA, USA) and 3,3′-diaminobenzidine tablets (D4293, Sigma-Aldrich, St. Louis, MO, USA) to visualize TH-positive neurons. The counting of total numbers of TH-positive neurons in the SN was conducted via an Optical Fractionator probe of Stereo Investigator software (MicroBrightfield, Williston, VT, USA).