Anti f4 80

Histologic and immunohistochemical analysis was performed on right kidney tissue from mice that underwent UUO or Sham surgery. The kidney tissues were fixed in 10% neutral buffered formalin and processed using standard techniques. 5 μm histological sections were prepared and stained with hematoxylin-eosin (H&E). Renal atrophy was semi quantitatively assessed as the percentage of cortical surface area occupied by atrophic tubules, compared to the entire cortical surface area, according to methods previously established in our laboratory [14 (link), 15 (link)]. Immunohistochemical staining was done for anti-F4/80 (1:200, BIO-RAD, Cat. No. MCA497RT), anti-KLF11 (1:1600, Novus Biological, Cat# H00008462-M03), anti-CD3 (1:100, Agilent Dako, Code Number A045), anti-CD68 (1:200, Abcam, Cat# ab125212), anti-CD163 (1:400, Abcam, Cat# ab182422), and anti-CD206 (1:800, Abcam, Cat# ab64693). Sections were stained with Sirius Red to quantitate matrix deposition. Ten random fields per kidney section were examined for each marker (F4/80, CD3, CD68, CD163, CD206 and Sirius Red) and the average of positive areas was expressed as percentage of total analyzed area. All measurements and quantifications were performed in a blinded fashion using NIS elements BR 4.13.00 64-bit image analysis system (Nikon Instruments INC., Melville, NY) at 200 X magnification.

Anti-amylase (sc-166349), anti-Ppar ɣ (sc-7273) and anti-cytokeratin 19 (sc-376126) antibodies were purchased from Santa Cruz Biotechnology; anti-Sox9 (#82630), anti-Pan-Keratin (C11; #4545), and anti-Mist1 (#14896) antibodies were purchased from Cell Signaling; anti-Ki67 (ab833) antibody was purchased from Abcam; anti CD45R (a B cell marker) was purchased from BioLegend (cat:103206) and anti F4/80 (a marker of mouse macrophages) antibody was from Bio-Rad. Anti-actin (clone AC-74) and anti-smooth muscle actin (SMA; clone 1A4) antibodies, tamoxifen, cerulein, azoxymethane (AOM), alcian blue 8GX, and safranin were purchased from Sigma. The heparanase inhibitors Roneparstat (SST0001) and Pixatimod (PG545) were kindly provided by Leadiant Biosciences S.p.A, Rome, Italy, and Zucero Therapeutics Ltd, Darra, Queensland, Australia, respectively [61 (link), 62 (link)]. A proteome profiler mouse cytokine antibody array (ARY028) was purchased from R&D systems.

Paraffin-embedded sections were de-paraffinized in xylene, rehydrated through graded alcohol, and immersed in PBS. The samples were pretreated with 0.4 mg/ml proteinase K (DAKO, Carpinteria, CA, USA) in a Tris-HCl (pH 7.6) buffer for 15 min at room temperature for antigen retrieval. Any residual enzymatic activity was removed by washing with PBS, and nonspecific staining was blocked by pre-incubation with PBS containing 10 % normal horse serum for 20 min at room temperature. Rabbit monoclonal anti-collagen type Ia1 and anti-collagen type IIa1 were purchased from Abcam (ab34710 and ab34712 Cambridge, MA, USA). Rat monoclonal anti-F4/80 was purchased from Bio-Rad (MCA497R Hercules, CA). Anti-collagen type I, type II collagen, and F4/80 (type I collagen 1:200 dilution; type II collagen 1:1000 dilution; F4/80 1:2000) were placed on each section for one hour at room temperature. After extensive washing with PBS, the sections were incubated in the secondary antibody of biotinylated anti-rabbit and rat IgG (Vector Laboratories, Burlingame, CA, USA) for 30 min at room temperature. Immunostaining was detected with VECTASTAIN ABC reagent (Vector Laboratories), followed by DAB staining. Counter staining was performed with Mayer’s hematoxylin.

BETi (ABBV-075, S8400; and ABBV-744, S8723) were purchased from Selleckchem (Houston, TX). CPI 203 (SML1212), phorbol 12-myristate 13-acetate (PMA) (P1585), and lipopolysaccharides (LPS) (L4391) were purchased from Sigma-Aldrich (St. Louis, MO). Bevacizumab (Genentech, South San Francisco, CA) was obtained from the MD Anderson Pharmacy and used for in vivo treatment. Interleukin 4 (IL-4) (200-04) and interleukin 13 (IL-13) (200-13) were purchased from PEPROTECH (Cranbury, NJ) and used for M2-like macrophage differentiation. For Western blot analysis, we used primary antibodies of anti-CCR2 (1:500, 711255, RRID: AB_2633142, Thermo Scientific, Waltham, MA), and anti-β-actin (1:1000, A5441, RRID: AB_476744, Sigma-Aldrich, St. Louis, MO). For immunohistochemical (IHC) staining, we used the following primary antibodies: anti-F4/80 (1:100, MCA497G, RRID: AB_872005, Bio-Rad, Hercules, CA), anti-Arginase-1 (ARG1) (1:200, 93668, RRID: AB_2800207, Cell Signaling, Danvers, MA), anti-Ki67 (1:100, RB9043P, RRID: AB_149874, Thermo Scientific, Waltham, MA), anti-CD31 (1:800, 553370, RRID: AB_394816, BD Biosciences, Franklin Lake, NJ), and anti-Cleaved PARP (Asp214) (D64E10) (1:50, 5625, RRID: AB_10699459, Cell Signaling, Danvers, MA).