Anti f4 80 antibody

Tissue slides were blocked with goat serum at room temperature for 15 min and incubated with anti-F4/80 antibody (Santa Cruz Biotechnology) at 4°C, overnight. Slides were washed in PBS and incubated with horseradish-peroxidase (HRP)-conjugated secondary antibodies for 30 min at 37°C. Staining was visualized by reaction with diaminobenzidine tetrahydrochloride (DAB) and counterstaining with hematoxylin, and stained sections were observed under a light microscope (DP73; Olympus).

Rapamycin, Three-methyladenine (3-MA), lipopolysaccharide (LPS), chloroquine (CQ), penicillin, streptomycin, and bovine serum albumin (BSA) were obtained from sigma (St. Louis., MO, US). IL-4 was purchased from R&D (Minneapolis, MN, US). Lysis buffer radioimmunoprecipitation assay (RIPA), phenylmethanesulfonyl fluoride (PMSF), and bicinchoninic acid (BCA) protein assay kit were obtained from Beyotime Institute of Biotechnology (Shanghai, China). Trizol reagent and cDNA reverse transcription kit were purchased from Invitrogen (Carlsbad, CA, US). The following antibodies were purchased: anti-LC3 antibody, S6K1 and pT389-S6K1 antibodies from Cell Signaling Technology (Danvers, MA, US), anti-Beclin1 antibody, anti-F4/80 antibody, and anti-IRF8 antibody from Santa Cruz (Dallas, TX, US), anti-AGEs antibody from Abcam (Cambridge, MA, US), β-actin antibody from Bioworld Technology (Louis, MN, US), CD206-APC, and CD11c-APC for flow cytometry from BD Biosciences (San Jose, CA, US). Alexa Fluor 488 conjugated goat anti-rabbit IgG second antibodies, and Alexa Fluor 594 conjugated goat anti-rat IgG second antibodies from Thermo Scientific (Hudson, NH, US).

DMEM/F-12 Medium, knockout serum replacement, glutamine, β-mercaptoethanol, nonessential amino acids, and human recombinant bFGF were purchased from Invitrogen Corporation (Carlsbad, CA, USA). EBM^TM-2 Basal Medium and EGM^TM-2 MV Microvascular Endothelial Cell Growth (EGM2-MV) Medium SingleQuots^TM supplements were purchased from Lonza (Basel, Switzerland). Trypsin-EDTA (0.25%) was purchased from Gibco (Waltham, MA, USA). Sodium chloride, 1-chloro-2,4-dinitrobenzene (DNCB), eosin Y, toluidine blue O, hematoxylin, and hydrochloric acid were purchased from Sigma-Aldrich (St. Louis, MO, USA). Total RNA Extraction Kit was purchased from iNtRON (Gyeonggi, Korea). High Capacity cDNA Reverse Transcription Kit was purchased from Thermo Fisher Scientific (Waltham, MA, USA). FastStart Essential DNA Green Master and serum-containing medium were purchased from Roche (Basel, Switzerland). Ammonia was purchased from JUNSEI (Chuo-ku, Tokyo). Anti-F4/80 antibody was purchased from Santa Cruz Biotechnology (Dallas, TX, USA). Mouse IgE ELISA Set was purchased from BD (Franklin Lakes, NJ, USA).

Paraffin embedded mouse kidneys were sectioned and stained with hematoxylin and eosin (H & E) or with PAS. Mean glomerular tuft volume was determined from the glomerular cross-sectional tuft area and mesangial matrix index was determined as described previously [26 (link)]. At least 20 glomeruli per mouse were scored from 4–5 mice per group. Immunohistochemistry was performed using anti-F4/80 antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA) and was detected using the HRPO-polymer (Biocare Medical).

To evaluate histopathological changes, the dorsal skin of each mouse was freshly excised and fixed with 10% neutral buffered formalin for 24 h. Tissues were processed using routine tissue techniques and embedded in paraffin. Paraffin-embedded specimens were sliced into 5-μm-thick sections. Sections were then transferred to adhesive microscope slides (Marienfeld, Lauda-Königshofen, Germany).
Deparaffinized skin sections were stained with hematoxylin and eosin (H&E) or toluidine blue for skin thickness measurement and mast cell detection. Additionally, immunohistochemical staining was conducted using an anti-F4/80 antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA). All stained sections were then examined with a light microscope Eclipse Ti (Nikon, Tokyo, Japan) to assess histological changes including skin thickening, mast cell infiltration, and F4/80-positive macrophage infiltration. Three sections per animal were used for histological examinations.

Dextran sodium sulfate was purchased from MP Biomedicals (Santa Ana, CA, USA), and hematoxylin and eosin solutions were obtained from Sigma Aldrich (St Louis, MO, USA). The ELISA kits for the detection of mouse tumor necrosis factor-alpha (TNF-α) and interleukin-6 (IL-6) were purchased from eBioscience (San Diego, CA, USA), and RIPA lysis buffer was obtained from Millipore (Darmstadt, Germany). Phosphatase and protease inhibitor cocktails were purchased from Roche (Basel, Swiss). A BCA protein quantification kit, fluorescence-tagged antibody and anti-Zonula occludens-1 (ZO-1) antibody were obtained from Thermo Fisher Scientific (Waltham, MA, USA), and anti-F4/80 antibody was purchased from Santa Cruz Biotechnology (Dallas, TX, USA).

All animal care and experiment procedures were conducted in accordance with the National Institutes of Health guidelines. The animal protocol was approved by the Institutional Animal Care and Use Committee of Washington University in St. Louis. To examine the in vivo toxicity of the hydrogel, 6 wt% hydrogel solution was subcutaneously injected into the 8-week-old C57BL/6 J mice. Prior to the injection, the pre-cooled hydrogel solution was sterilized under UV light for 30 min. As controls, mice injected with collagen gel were used. After seven days, tissue specimens were harvested at the injection sites and fixed with 4% paraformaldehyde for 24 h. The tissue sections (5 µm thick) were stained with anti-F4/80 antibody (Santa Cruz) and DAPI. The stained sections were imaged with an Olympus FV1200 confocal microscope. The F4/80+ cell ratio was quantified by normalizing F4/80+ cell number to the total cell number in each image.