Anti mouse or anti rabbit biotinylated secondary antibody

We performed IHC as described previously by Awaji et al. [47 (link)]. In brief, we incubated slides with primary antibodies, such as αPlexin-B3 (Santa Cruz-sc671441; 1:100), αKi67 (1:100; Santa Cruz-sc15402), αALDH1-A1(1:100; sc374149), αCD31 (1:50, Abcam,-ab28364), and αCD44 (1:500; Abcam-ab157107), in antibody diluent overnight at 4 °C. The next morning, we washed the slides and incubated with biotinylated anti-rabbit or anti-mouse secondary antibody (Vector Laboratories, Burlingame, CA, USA) for 45 min. Images were captured through the Nikon microscope using NIS Element software.
IHC scoring was performed according to the following criteria: percentage of positive cells on the slides was as follows: 0 (negative), 0.1 (1–10% of cells positive), 0.2 (11–20% of cells positive), and 0.3 (20–30% of cells positive), and so forth. Furthermore, the intensity was designated as weak (1 point), moderate (2 points), strong (3 points), or very strong (4). The IHC composite score was calculated by multiplying the extent of positive cells with intensity. Two independent observers examined each slide, and their observations were positively correlated with each other. Average scores were used for analysis, and if the two observers significantly differed in their scoring, a third observer examined the slide.

We performed immunohistochemistry as described previously [11 ]. In brief, we incubated slides with αPCNA, (1:100; Santa Cruz, CA); or αSEMA5A [5 (link)] (1:50) primary antibodies in antibody diluent overnight at 4 °C. Next morning, we washed the slides and incubated with biotinylated anti-rabbit or anti-mouse secondary antibody (Vector Laboratories, Burlingame, CA) for 45 min. We quantitated the cell number for a particular stain by counting at least five different random fields in the same section at high resolution using a Nikon microscope.

RA, OA, and normal (NL) (no arthritis) ST cryosections as well as ankle sections of Wt mice induced with K/BxN serum were fixed in cold acetone for 30 min at 4 °C. The tissue sections were blocked with 5 % donkey serum and 20 % fetal bovine serum (FBS) in phosphate-buffered saline (PBS) at 37 °C for 1 h. The sections were then incubated with either mouse anti-human Id1 antibody (Abcam, Cambridge, MA, USA, 10 μg/mL), rabbit anti-mouse Id1 antibody (CalBioreagents, San Mateo, CA, USA, 10 μg/mL), or purified nonspecific mouse and rabbit immunoglobulin G (IgG) (Thermo Fisher Scientific, Waltham, MA, USA) for 1 h at 37 °C in blocking buffer. After washing, tissues were incubated with a biotinylated anti-mouse or anti-rabbit secondary antibody (Vector Laboratories, Burlingame, CA, USA, 10 μg/mL) for 1 h at 37 °C in blocking buffer. Vectastain ABC kit (Vector Laboratories) was used to detect the antibodies on the tissues, following manufacturer’s protocols. Sections were mounted with Cytoseal 60 (Thermo Fisher Scientific), visualized under an Olympus microscope (Olympus, Tokyo, Japan) and scored by a pathologist.

Standard Western blot procedures were carried out with the following antibodies: anti-acrolein (1:1 000; Abcam, Cambridge, MA; Cat. No.) and anti-α-syn (1:500; Abcam, Cat. No. ab27766). Briefly, brain tissues were sonicated in 1x RIPA buffer with protease inhibitor. After centrifugation, the supernatant was collected for Western blotting. Sixty micrograms of protein with 20% SDS, β-mercaptoethanol, and 2x Laemmli buffer were loaded onto a 15% Tris-HCL gel and electrophoresed at 80 V for 2–3 h. The proteins were then transferred to a nitrocellulose membrane by electro-blotting at 70 V for 1–2 h (depending on the protein size) at 4 °C in 1x transfer buffer with 20% methanol. The membrane was blocked in 1x casein (Vector) at room temperature for 1 h, and immunolabeled with the primary antibody overnight at 4 °C. The membrane was further incubated with biotinylated anti-mouse or anti-rabbit secondary antibody (Vector) at room temperature for 1 h. The DuoLux substrate (Vector) immunodetection kit was used for chemiluminescent signal, and imaged using a Western blot imager (Azure Biosystems, Dublin, CA). The AlphaView software (Protein Simple, San Jose, CA) was used to quantify the relative signals for each band. Data were normalized with actin and are expressed as the percent of control.