Ab283654

Immunohistochemical staining of the paraffin-embedded tissues was performed as follows: Following deparaffinization and antigen retrieval, the kidney tissue sections were blocked with 10% goat serum for 1 h and then incubated overnight at 4°C with the following antibodies: Anti-CD68 antibody (1:100, cat. no. ab283654; Abcam) and anti-lymphocyte antigen 6 complex, locus G (Ly-6G) (1:200, ab25377; Abcam). The following day, following incubation with HRP-conjugated secondary antibodies (1:500, cat. no. ab97057; Abcam; 1:500, cat. no. 7074, Cell Signaling Technology, Inc.) for 1 h at room temperature, the slides were developed with DAB until the signal clearly appeared, and the nuclei were stained with hematoxylin for 5 min at room temperature, and images were obtained using a microscope (Olympus IX83, Olympus Corporation).

Sections were de-paraffinized and hydrated. Antigen retrieval was performed in a decloaking chamber (Biocare Medical, Pacheco, CA, USA) using citrate buffer pH 6.0. Endogenous peroxidases and non-specific binding sites were blocked with Bloxall (Vector Laboratories, Burlingame, CA, USA), rodent block (Biocare Medical) and horse serum (10%). Primary antibodies anti-NR2F6/Ear2 (ab137496, Abcam, Waltham, MA, USA, 1:3000 dilution), anti-GPNMB (ab188222, Abcam, Waltham, MA, USA, 1:1000 dilution) were added overnight. An HRP-conjugated horse anti-rabbit IgG Polymer (Vector Laboratories) was used as secondary for 30 min and the signal was developed using the SignalStain DAB Substrate Kit (Cell Signaling). For IF, anti-CD68 (ab283654, Abcam, Waltham, MA, USA, 1:1000 dilution) and anti-CD72 (AF1279, R&D Systems, Minneapolis, MN, USA, 1:40 dilution) antibodies were coadministered overnight and were labeled with horse anti-rabbit or anti-goat Dylight 488 and 594 (Vector Laboratories, 1:300 dilution) secondary antibodies for 30 min. Alternatively, an Opal kit (Akoya Biosciences, Marlborough, MA, USA) was used with anti-NR2F6/Ear2 (ab137496, Abcam, Waltham, MA, USA, 1:700 dilution) and anti-CD206/Mrc1 (24595T, Cell Signaling, Danvers, MA, USA, 1:500 dilution) primary antibodies.