Rabbit anti mouse cd68 antibody

Paraffin embedded tissues were analyzed using immunohistochemical staining [49 (link)] with the following primary antibodies: anti-CD68 antibody (DAKO, Carpinteria, CA), anti-α-SMA antibody (DAKO, Carpinteria, CA), anti-CCL7 antibody (Gen Way Biotech, San Diego, CA), anti-COL3A1 antibody (Bioss, Beijing, China), anti-FOXP3 antibody (Biolegend, San Diego, CA) and rabbit anti-Mouse CD68 antibody (Bioss, Beijing, China). For quantification of tumor stromal cells within the tumor area, CD68 was used as a pan-macrophage marker, α-SMA was used to detect cancer activated fibroblasts adjacent to RCC cells [50 (link)], FOXP3 was used as a specific marker for regulatory T cells (Tregs) [22 (link)], and mast cells were assessed using the routine toluidine blue staining method [21 (link)]. Each tumor section was evaluated by using 20× objective lens, and five independent areas with the most abundant positive cells were selected, digitally photographed, and manually counted under a microscope. The average positive cell counts for each patient were used for statistical analysis. For quantification of CD68⁺ cells in CDX xenografts, four sections from each xenograft were randomly selected and quantified as described above, the average positive cells for each mouse were used for statistical analysis. Results were confirmed by two pathologists in a double-blind analysis.

All treatment and analysis reagents and biochemical kits utilized in this study were obtained from commercial sources: Cholesterol quantification kit, simvastatin, progesterone, nile red, filipin and 7β‐Hydroxycholesterol (Sigma‐Aldrich, St. Louis, MO, USA); anti‐NPC1 antibody (EMD Millipore, Billerica, MA, USA); Mouse interleukin (IL)‐1 beta/IL‐1F2 Quantikine ELISA kit (R&D Systems, Minneapolis, MN, USA); GenMute^™ siRNA Transfection Reagent (SignaGen Laboratories, Gaithersburg, MD, USA); oxLDL (TBARS: 29‐44 nmoles MDA/mg; Alfa Aesar, Ward Hill, MA, USA); rabbit anti‐mouse CD68 antibody (Bioss, Woburn, MA, USA); Alexa fluor 633 goat anti‐rat IgG (Life Technologies, Carlsbad, CA, USA); LXRα siRNA, NPC1 siRNA and LAMP‐1 rat monoclonal antibody (Santa Cruz Biotechnology, Dallas, TX, USA); cytochrome P450 7A1 siRNA (Origene, Rockville, MD, USA); solid‐phase extraction (SPE) column (Silicycle, Quebec, QC, Canada); 22(S)‐hydroxycholesterol (D7) (Avanti Polar Lipids, Alabaster, AL, USA); HPLC column, XTerra RP18, 2.1 × 150 mm, 5 μM (Waters Corporation, Milford, MS, USA); and lysosome enrichment kit (Thermo Scientific, Waltham, MA, USA). C57BL/6J mice were obtained from the Jackson Laboratory (Bar harbor, ME, USA) and cared for under housing and animal protocols approved by the Institutional Animal Care and Use Committee at Virginia Commonwealth University.