3 3n diaminobenzidine tertrahydrochloride dab

For immunohistochemistry, ovarian tissue were fixed in 4% paraformaldehyde and embedded in paraffin. 5-μm-thickness sections were dewaxed, rehydrated and performed heat mediated antigen retrieval in Citrate buffer for 15 minutes. After blocking the endogenous peroxidase by 3% hydrogen peroxide and unmasking the antigen by 5% goat serum, the sections were incubated with primary antibodies for 3 hours at room temperature. After washing, sections were incubated with 1:500 diluted horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG (H+L) (111-035-003, Jackson Immuno-Research Laboratories) for 1 hour at room temperature. Sections were incubated with 3,3N-Diaminobenzidine Tertrahydrochloride (DAB, Beyotime) to detect the peroxidase activity and mounted with aqueous mounting medium (Dako). The primary antibodies were rabbit anti-GDF-9 antibody (BS-175R, Beijing Biosynthesis Biotechnology, 1:500 dilution) and rabbit anti-PCNA antibody (24036-1-AP, Proteintech, 1:200 dilution).

F4/80, also called as EGF-like module-containing mucin-like hormone receptor-like 1 (EMR1), is a heavily glycosylated G-protein-coupled receptor and is a well-known biomarker for mouse macrophages [72 (link)]. F4/80 was detected by immunohistochemistry (IHC). Briefly, lung sections (5 μm) were mounted onto the slides, deparaffinized and rehydrated, and heated in 0.1 M citrate buffer (pH 5.8) for antigen retrieval. To inactive endogenous peroxidase, we incubated slides with 3% H₂O₂ at RT for 15 min. After blocking with 2% BSA for 30 min at 37 °C, the slides were incubated with primary antibodies against F4/80 (#30325; Cell Signaling Technology, MA, USA) overnight at 4 °C. After incubation with the corresponding secondary antibody for an additional 1 h at room temperature in dark, the slides incubated in 10 μg/ml 3, 3 N-diaminobenzidine tertrahydrochloride (DAB; Beyotime Biotech Inc., Nantong, China) for 10 min. The nuclei were counterstained with hematoxylin for 10 s. The monocytes (THP-1) treated with 100 ng/ml phorbol myristate acetate for 48 h were regarded as a positive control. Positive staining cells were counted in 20 random fields per section in each group by light microscopy (Leica, Germany) and the average number of positive cells per section was calculated.

Immunohistochemistry was used to analyze the expression of PINK1 in lung tissue. The sections underwent xylene deparaffinization, rehydration in graded alcohol solutions, and heat pretreatment for antigen repair. After quenching endogenous peroxidase and blocking non-specific proteins, sections were exposed to PINK1 antibody for an overnight incubation at 4°C. Finally, the sections were treated with a secondary antibody that was conjugated to HRP and stained with 3,3N-Diaminobenzidine Tertrahydrochloride (DAB, Beyotime Biotechnology, China). The images were observed at ×200 magnification under a light microscope.