Anti cd8

Formalin-fixed paraffin-embedded samples were sectioned into 4-μm slices. Deparaffinized sections were subjected to antigen retrieval with citrate solution, pH 6.0, and treated with 3% hydrogen peroxide to inhibit endogenous peroxidase activity. After blocking slides with FBS, sections were incubated overnight at 4°C with a 1:200 dilution of anti-vascular endothelial growth factor (VEGF) (Abcam, ab1316), 1:100 dilution of anti-VCAM-1 (Santa Cruz Biotechnology, sc-1504), anti-CD4 (Abcam, ab51312), and anti-CD8 (Santa Cruz Biotechnology, sc-7970) monoclonal antibody. Slides were treated with biotin-conjugated secondary antibody (Dako) and with horseradish peroxidase-conjugated avidin (Dako). Peroxidase activity was localized for all samples with 3,3′-diaminobenzidine, counterstained with hematoxylin, dehydrated, cleared, and mounted in mounting medium Entellan (Merck).

On day 5 after the operation, the heart grafts were harvested. After paraformaldehyde fixation and paraffin-embedding, the heart grafts were cut into paraffin sections, stained with HE, and observed by light microscopy (BX51, Olympus, Japan). The standards published by the International Society for Heart and Lung Transplantation were used to evaluate the grades of the rejection response [29 (link)]. For immunofluorescence staining, the sections were dewaxed, restored by microwave heating, blocked with 5% goat serum (Beyotime Bio, #C0265, China), and then incubated with anti-CD4 (1 : 100, Santa Cruz, #sc-20079, China) and anti-CD8 (1 : 100, Santa Cruz, #sc-1177, China) overnight at 4°C. The next day, the sections were incubated with CY3-labeled goat anti-mouse secondary antibodies (1 : 50, Servicebio, #GB22301, China) and subsequently stained with 4′,6-diamidino-2-phenylindole (DAPI, 2 μg/ml; Servicebio, #G1012-10ML, China) for five minutes. The images were acquired by fluorescence microscopy (Olympus BX6 with a DP72 Camera, Japan) and then analyzed by the ImageJ software.

Unstained sections were incubated with one of the next primary mouse monoclonal antibodies: anti-CD4 or anti-CD8 (for mature lymphocytes) or MPO (neutrophils) (1:100, Santa Cruz Biotechnology) in a humidified chamber at 37 °C overnight. Sections were washed with PBS and were incubated with secondary antibody with fluorescein isothiocyanate-conjugated anti-mouse Alexa Fluor 488 (1:2000, Molecular Probes, Life Technologies) for 1 h at 37 °C. Sections were rinsed and nuclear counterstain with 2 μg ml⁻¹ DAPI in PBS was added. Sections were observed using a Leica DM2000 microscope. Each microscopic field was acquired at 20 × magnification to maximize signal and analysed.