Mayer s hematoxylin

Four micrometers of formaldehyde‐fixed pleural tissue were dewaxed and rehydrated with an alcohol gradient and PBS. Antigen retrieval was performed with citrate (pH 6.0). Endogenous peroxidase was blocked with 3% H₂O₂ in water for 20 min, and nonspecific binding was blocked with diluted normal goat serum for 60 min, and was then incubated with the primary antibody (mouse anti‐human monoclonal antibody C1q (ab71089), rabbit anti‐human polyclonal antibody factor B (ab192577), rabbit anti‐human polyclonal antibody factor P (ab186834), mouse anti‐human monoclonal antibody MBL (ab23457), mouse anti‐human monoclonal antibody factor H (ab118820), mouse anti‐human monoclonal antibody C3a (ab37230), mouse anti‐human monoclonal antibody C5a (ab11877) and rabbit anti‐human polyclonal antibody SC5b‐9(ab55811), all antibodies were purchased from Abcam) for 18 h at 4°C. According to the manufacturer's instructions, labeling was identified using the SP goat IgG kit (PV‐6000, ZSGB‐Bio, China). The chromogenic reaction solution contained 3, 3′‐diaminobenzidine (DAB) (ZLI‐9018, ZSGB‐Bio, China), and counterstaining was performed with Mayer's hematoxylin (Solarbio). Slides were viewed under an imaging fluorescence microscope (Olympus BX51; Olympus).

Deparaffinization and rehydration of cardiac tissue sections were reached in dimethylbenzene and graded ethanol, followed by treatment of sections with 3% H₂O₂ for 10 min. The sections were heated with antigen retrieval buffers to repair antigen for 10 min at 95 °C and cooled to room temperature. Sections were blocked with 5% bovine serum albumin at 37 °C for 1.5 h. After 3 times' washes, sections were incubated with anti-p16 antibody (Affinity Biosciences, Cat# AF-0288, 1:250) at 4 °C overnight. The next day, sections were incubated with the HRP conjugated secondary antibodies (ZsBio, Beijing, China) at room temperature for 1 h and colored by DAB (ZsBio). Nucleus were stained with Mayers hematoxylin from Solarbio (Beijing, China).