Ab100790

Frozen sections of LC tissues and normal tissues were rewarmed, fixed in ice-cold acetone, reacted with 0.3% tritonX-100 and blocked with 5% bovine serum albumin. After that, the sections were successively probed with the primary antibody F4/80 (Ab100790, 1:100, Abcam, Cambridge, UK) and horseradish peroxidase-labeled goat anti-rabbit immunoglobulin G antibody (ab6721, 1:1000, Abcam) and developed by diaminobenzidine. Then, the sections were reacted with hematoxylin, dehydrated, and permeabilized. Positive expression was reflected by brown particles. The sections were viewed under five fields of view under a microscope [17 (link)].

Tumor tissues collected from each group of mice were fixed in 4.0% paraformaldehyde, embedded in paraffin and cut into 4-μm sections. H&E staining was performed and used to measure the tumor burden. On the other hand, tumor sections were incubated with 3.0% hydrogen peroxide to inactivate endogenous peroxidase. After antigen retrieval, the sections were blocked in 5.0% bovine serum albumin and incubated with the following primary antibodies at 4 °C overnight: rabbit anti-mouse F4/80 (ab100790; Abcam, Cambridge, MA, USA), CD31 (ab28364, Abcam) and Ki67 (ab16667, Abcam). After washing with PBS, further incubation with a biotin-conjugated secondary antibody was performed, followed by visualization with 3,3′-diaminobenzidine (DAB) and counterstaining with hematoxylin for examination on an inverted microscope (IX71, Olympus, Tokyo, Japan).

The tumor tissues were fixed with 4% formaldehyde at 4 ˚C overnight and then embedded with paraffin. The tissues were sectioned into 4–5 µm slices. For hematoxylin and eosin (HE) staining, the dewaxed sections were stained with hematoxylin (Sigma, St. Louis, MO, USA) for 15 Min, and then incubated in hydrochloric acid alcohol (1%) for 15 Sec. After soaking in eosin (Sigma) for 2 Min, the slices were observed under an optical microscope. For theimmunohistochemical (IHC) assay, the sections were stained with F4/80 macrophage marker, EZH2, CCL5, or MMP2 (1:100, ab100790; Abcam, USA). In brief, after deparaffinized with xylene, the sections were subject to retrieval in a heating citrate buffer (pH 6.0) for 15 Min. After cooling down to temperature, the sections were blocked with normal goat serum (Thermofisher) at 37 ˚C for 10 Min and then incubated with primary anti‐F4/80, EZH2, CCL5, or MMP2 antibodies overnight at 4 °C. Next, sections were incubated with anti‐rabbit secondary antibody (biotinylated) (1:1,000, ab6720, Abcam) at room temperature for 30 Min. The horseradish peroxidase‐conjugated streptavidin (Amersham, Amersham, UK) was incubated with sections at 37 ˚C for 30 Min. The diaminobenzidine (Sigma) was used for chromogenic detection. The staining was observed under an inverted microscope (Olympus, Tokyo, Japan) (400× magnification).

After blocking with 10% goat serum for 1 h, macrophages were identified using a rabbit polyclonal antibody for F4/80 (Abcam ab100790, Cambridge, MA) at 1:300 dilution overnight at 4°C and AlexaFluor546 anti-rat secondary antibody (Invitrogen) at 1:400 dilution for 2 h at room temperature. Ceramide was stained using a mouse monoclonal antibody (clone MID 15B4) at 1:100 dilution overnight at 4°C and AlexaFluor488 anti-mouse secondary antibody (Invitrogen) at 1:400 dilution for 2 h at room temperature. All antibody labeling was carried out in PBS containing 3% goat serum. Images were acquired with a Zeiss LSM 510 laser-scanning confocal microscope (Thornwood, NY) using a 63 × 1.4 NA objective.

For immunohistochemistry, both flank tumors were dissected from the immunocompetent mice and formalin-fixed and paraffin-embedded (FFPE) on day 14 after grouping. Four-micrometer-thick tissue sections were prepared and used for IHC staining. IHC was carried out according to a standard protocol. The following antibodies were used for the IHC experiment: CD8 antibody (Affinity, AF5126, China), CD16 (Affinity, DF7007, China), and F4/80 (Abcam, ab100790, USA).

Antigen retrieval was performed using 20–40 µg/ml proteinase K (Sigma) digestion at 37°C for 15 min. The sections were then blocked with 10% goat serum and 0.5% bovine serum albumin (BSA) in 3% milk for 1 h at room temperature, followed by primary antibody labeling, mouse–antimouse F4/80 (1:100, ab100790, Abcam) or rabbit–antimouse erythropoietin receptor (EPOR, 1:800, PAB18350, Abnova) at 4°C overnight. The next day, DAKO secondary (K4063) was applied to the sections for 30 min. The antibody binding was then revealed by 3,3′-diaminobenzidine (DAB, Vector, Burlingame, USA) and hematoxylin counterstaining.
For double labeling of properdin and F4/80 in the kidneys of WT mice, proteinase K at 40 µg/ml was used for antigen retrieval at 37°C for 30 min. After the staining of F4/80 was obtained, the sections were then incubated with primary antibody rabbit–antimouse properdin (1:200, ABF185, Merck Millipore) at 4°C overnight. Afterwards, biotinylated secondary goat–antirabbit immunoglobulin G (IgG) was applied to the slides for 30 min at 37°C (1:300, BA-1000, Vector), followed by alkaline phosphatase streptavidin (1:200, SA-5100, Vector) for 30 min at 37°C, and then developed by Fast Red (Sigma) for 8 min.

To identify the intragraft infiltration of CD4⁺ and CD8⁺ cells, specific markers were stained for grafts (n = 10 for each group). Briefly, 0.01 mol/L sodium citrate buffer was used on tissue sections for antigen repair. The endogenous peroxidase was eliminated through incubation with 3% hydrogen peroxide for 25 min. Then the sections were incubated with rabbit anti-mouse CD4 antibody (diluted at 1:1,000, ab183685, Abcam, Cambridge, UK), rabbit anti-mouse F4/80 antibody (diluted at 1:1,000, ab100790, Abcam, Cambridge, UK), rabbit anti-mouse CD8 antibody (diluted at 1:2,000, ab217344, Abcam, Cambridge, UK), or rabbit anti-mouse Foxp3 antibody (diluted at 1:1,000, ab215206, Abcam, Cambridge, UK), overnight at 4°C and then incubated with 100 μl enhanced enzyme-labeled goat anti-rabbit IgG polymer (DAB kit, PV9000, ZSGB-BIO, Beijing, China) for 20 min next day at room temperature. Finally, a freshly prepared DAB solution was used to visualize the labeling. Non-specific staining was determined according to the negative control. The ImageJ (version 1.53, National Institutes of Health, USA) software was applied to quantify different cell types infiltration.