D2s9r

For detection of immune cells and phagocytes in capsule, histochemistry and immunohistochemistry (IHC) analysis were performed. The Periodic Acid Schiff (PAS) staining was performed to identify the leukocytes-granulocytes infiltrated in capsular tissue. Toluidine blue (1%, pH 4.0) staining was performed to identify mast cells in tissue sections through metachromatically reaction of heparin and sulfated glycosaminoglycan granules. For IHC detection of phagocytes, histological sections were submitted to reaction with the primary antibody F4/80 (rabbit monoclonal, 1:100, D2S9R, #70,076, Cell Signaling, Danvers, MA, USA). Slides were dewaxed, and antigens were retrieved in 10 mM citrate buffer (pH 6.0, at 98 °C). Endogen peroxidases blocked in 10% H₂O₂, and nonspecific proteins were blocked using 5% skimmed milk. After, they were incubated overnight with the primary antibodies. An incubation with polymer (Novolink Max Polymer DS (1250), Leica) was performed, and detection was performed with 3–30′-diaminobenzidine tetrahydrochloride solution (DAB) and counter-stained with Harris Hematoxylin, staining and inspected under a light microscope (Olympus BX50, Olympus Corporation, Center Valley, PA, USA). Images were captured using Olympus cellSens Standard 1.18 software (Olympus Corporation, Center Valley, PA, USA).

Paraffin slides were used for immunohistochemistry (IHC) for F4/80 (a marker of macrophages). First, the paraffin slides were baked at 60 °C overnight and then deparaffinized by xylene and rehydrated with ethanol. Sodium citrate antigen-retrieval solution (Beyotime, Shanghai, China) was used for retrieval with a low-level microwave. Hydrogen peroxide (1.5%) in methanol was used to block endogenous peroxidase activity, and Dako protein-blocking reagent (DakoCytomation Corporation, Carpinteria, CA, USA) was used to block the irrelevant antigen. The primary Ab F4/80 (D2S9R, working concentration of 1:500, Cell Signaling Technology, Danvers, MA, USA) was incubated overnight at 4 °C. For isotype control slides, normal rabbit IgG (Calbiochem, San Diego, CA, USA) was used as the primary Ab. A Dako Real Detection System Alkaline Phosphatase Kit (K5005, Dako) and chromogen (Fast Red) were used for immunostaining. Hematoxylin was used to counterstain, and the slides were then dehydrated.