Rat anti mouse cd68

The xenograft tissues (n = 3 for each group) were fixed with 10% formalin and embedded in paraffin. The paraffinized tissues were cut into 2-μm sections and placed on frontier micro slide glass. The sections were deparaffinized by 99.9% xylene, following 100%, 95%, 70% ethanol and finally distilled water. The sections were fixed with 4% paraformaldehyde, permeabilized in 0.2% trition in PBS, then blocked with 5% NGS (normal goat serum) for 2 hour, followed by incubating with a primary antibody, which was used at a final concentration of 5-10 μg/ml in 2% NGS and included: anti-mouse endothelial cell (MECA-32, BioLegend, California, USA), mouse anti-rat CD68 (AbD Serotec, Kidlington, UK), anti-CD83 (Michel-19, BioLegend), aand anti-mouse CD8 (BioLegend, California, USA). Sections were incubated overnight with primary antibody at 4°C and then incubated with appropriate horseradish peroxidase-conjugated secondary antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA) after washing with 0.1% Trition in PBS. The sections were finally mounted using DAPI Fluoromount-G (SouthernBiotech, Birmingham, USA). Fluorescent images were captured by Olympus microscope BX53 using MetaVue software under the same conditions (10 or 1,000 ms exposure time, high & low limits, and scaling) and the signal intensity was analyzed with the threshold images that excluded the background signal.

CD68 expression was used to assess the purity of cell populations prior to array analysis. Cells were harvested using a cell scraper and washed with PBS containing 1% bovine serum albumin (BSA). Cells were permeabilised using Leucoperm (AbD Serotec) according to instructions and incubated with mouse anti-rat CD68 (AbD Serotec, clone ED1, 1:200) followed by goat anti-mouse IgG APC (BioLegend, 1:400) and analysed on a FACSCalibur (BD). Data was analysed using FlowJo (Tree Star). Quadrants were set using the mouse IgG1 isotype control (AbD Serotec, clone F8-11-13).

To evaluate the presence of neuroinflammation after peripheral LPS treatment, prefrontal coronal sections were tagged for glial fibrillary acidic protein (GFAP) or ionized calcium binding adaptor molecule (Iba-1) costained with CD68 as astrocyte and microglial markers, respectively. Free-floating sections were rinsed of cryoprotectant solution, incubated in antigen unmasking solution (Vector, Burlingame, CA, USA) at 95°C for 7 min, and rinsed 3 × 5 min in 1× PBS. Sections were blocked in 10% donkey serum, 0.3% Triton, and PBS for 2 h and incubated overnight in primary antibody solution, including 1% bovine serum albumin (BSA), 0.3% triton, and PBS (chicken anti-GFAP: 1:500, ABCam, Cambridge, MA, USA; goat anti-Iba-1: 1:500, ABCam; mouse anti-rat CD68: 1:200, BioRad, Hercules, CA, USA). The sections were rinsed and incubated for 2 h in secondary antibody solution of 1% BSA in PBS (Alexa Fluor 488 donkey anti-chicken, 1:500, Jackson, Bar Harbor, ME, USA; Alexa Fluor 488 donkey anti-goat, 1:500, Life Technologies, Waltham, MA, USA; Alexa Fluor 594 donkey anti-mouse, 1:800, Life Technologies, Waltham, MA, USA). After a final rinse, slices were mounted onto gelatin-coated slides and coverslipped with mounting medium containing DAPI (Vector).

The kidney samples were washed in cold PBS and fixed in 4% phosphate-buffered formaldehyde overnight at 4°C. Then, the samples were cryoprotected in 1.1 M sucrose, embedded in OCT Cryomount (Histolab Products AB, Sweden; 45830), and stored at –76°C. Frozen kidney sections (8 µm) were prepared on adhesive slides, rehydrated, and incubated with 50 mM NH₄Cl for 30 min. Thereafter, sections were permeabilized with 0.25% Triton X-100 in PBS for 10 min and blocked with 5% goat serum in PBS for 1 h at room temperature. To identify macrophages, sections were stained with mouse anti-rat CD68 (Bio-Rad, USA; 1:100 dilution; MCA341R) overnight at 4°C. Next, the sections were incubated with an Alexa Fluor 660-conjugated secondary antibody (Thermo Fisher Scientific; 1:750 dilution; A-21055) for 1 h at room temperature. Cell nuclei were counterstained with DAPI (Roche, USA; 1:10,000 dilution; 10236276001). The CD68-positive cells were analyzed using a Zeiss Axioscope (Carl Zeiss, Germany) fluorescence microscope. The cells were counted in 30 randomly selected images of the renal cortex and averaged from 2 sections per individual. The cell numbers were calculated per mm².

For analyses inflammatory cell distribution, OCT‐embedded tissue sections were fixed with 4% PFA and stained with the following primary antibodies for confocal microscopy: rabbit anti‐rat α‐actinin (Abcam), mouse anti‐rat CD68 (Bio‐Rad). The appropriate fluorescently conjugated secondary antibodies (Invitrogen) were applied prior to mounting using Fluoroshield with DAPI (Sigma‐Aldrich).