Biotinylated anti rabbit igg

Following deparaffinisation and a brief rinse in phosphate-buffered saline (PBS), sections were boiled in 10 mM Tris-EDTA solution (pH 9.0) for 40 min for antigen retrieval. After rinsing with PBS, the sections were incubated in blocking solution containing 5% normal swine serum (NSS), 5% bovine serum albumin (BSA), and 0.25% carrageenan in PBS for 2 h. The sections were processed for immunohistochemistry with primary antibodies against PSAP (IM-1), GPR37, and GPR37L1 at a concentration of 1 μg/ml, and incubated overnight at 4°C. The sections were then rinsed with PBS and incubated with biotinylated anti-rabbit IgG (1:500; Dako, Glostrup, Denmark) for 3 h 30 min at 32°C. After rinsing again with PBS, the sections were incubated with the avidin-biotin complex (ABC) and visualised using a VECTASTAIN ABC kit (Vector Laboratories, Burlingame, CA, USA) for 3 h 30 min at 32°C. Finally, the sections were rinsed again with PBS, and the colour reaction was developed using the diaminobenzidine (DAB) method.

For the immunohistochemistry and immunofluorescence of GPR37 and GPR37L1, boiling for antigen retrieval is necessary, but it is not necessary for PS. Following deparaffinization, brain sections were boiled for 20 min and cooled in 10 mM sodium citrate, after which the sections were exposed for 2 h to a blocking solution containing 1% normal goat serum, 5% bovine serum albumin, 0.2% fish gelatin, 0.1% triton X, and 0.1% NaN₃ in PBS. The sections were processed for immunohistochemistry with anti-PS (IM-1), anti-GPR37, and anti-GPR37L1 at a concentration of 1 μg/mL overnight at 4°C. The sections were then rinsed with PBS and incubated with biotinylated anti-rabbit IgG (Dako) for 2 h at 32°C. After rinsing again with PBS, the sections were incubated for 30 min at 32°C with peroxidase-conjugated streptavidin (Vector). The sections were rinsed with PBS and subjected to a color reaction with diaminobenzidine (DAB).

For immunohistochemistry analysis, ovaries were obtained from mice (n = 5 mice per group) sacrificed at 24 h after Cy or PBS treatment. After deparaffinisation of the ovaries in xylene and rehydration by graduated ethanol washes, endogenous peroxidase activity in the sections was inactivated by 3% (vol/vol) hydrogen peroxide in PBS. After epitope retrieval (at 98 °C for 30 min in 0.01 M citrate buffer solution, pH 6.0; Dako, Glostrup, Denmark), non-specific binding was blocked with 2% bovine serum albumin for 1 h at room temperature. The sections were incubated with primary antibody overnight at 4 °C. Antibodies for Foxo3a (1:1000; 12829S, Cell Signaling Technology, Danvers, MA, USA), Ki67 (1:400; 12202S, Cell Signaling Technology), and cleaved caspase-3 (1:200; 9661S, Cell Signaling Technology) were used. After washing, slides were incubated with biotinylated anti-rabbit IgG (Dako) for 1 h. Finally, protein signals were visualised by DAB (Dako) staining. After stopping the reaction with distilled water, the slides were counterstained with haematoxylin, dehydrated, and mounted. Images were obtained using a digital camera (Olympus, Tokyo, Japan) mounted on a conventional light microscope (Olympus) at a magnification of × 40 [26 (link)].

Formalin-fixed, paraffin-embedded tissue Sections (8 µm) from the frontal and occipital cortices were cut from cases 1–6 listed in Table 1. Sections were deparaffinised in xylene and rehydrated using graded alcohols. Immunohistochemistry for all antibodies required pre-treatment with a pressure cooker for 10 min in citrate buffer pH 6.0. Endogenous peroxidase activity was blocked in 0.3% H₂O₂ in methanol for 10 min and non-specific binding with 10% dried milk solution. Tissue sections were incubated with primary antibodies; MRC1 (1:1000; R&D systems), PROX1 (1:400; Acris), LYVE1 (1:50; Abcam), LYVE1(1:200; R&D Systems), PDPN (1:350; Sigma-Aldrich), and VEGFR3 (1:40; R&D Systems) for 1 h at RT, followed by biotinylated anti-rabbit IgG (1:200; Dako) or biotinylated anti-mouse IgG (1:200; Dako) for 30 min at RT and Avidin–Biotin complex (30 min; Dako). Colour was developed with di-aminobenzidine/H₂O₂ [32 (link)].
Images were acquired using a Nikon Eclipse Ni microscope using 10×, 20×, and 40× air objectives and 60× oil objective.

Paraffin sections with a thickness of 3 µm were processed for immunohistochemistry. For labeling with primary antibodies, the sections were deparaffinized and treated for 30 min with 0.3% H₂O₂ in methanol. All the sections were heated in an autoclave for 20 min in citrate buffer (pH 6.0), then incubated with the primary antibodies overnight. The primary antibody and conditions used were as follows; anti-p21^Waf1/Cip1 (12D1)(1:50 rabbit monoclonal, #2947, Cell Signaling Technology). Sections were stained with biotinylated anti-rabbit IgG (Dako, CA, USA) and visualized using H₂O₂-containing diaminobenzidine buffer. The anti-p21^CIP1 rabbit monoclonal antibody was validated as described above, in 2.8 Immunocytochemistry analysis section.