Anti goat hrp dab cell and tissue staining kit

Ovaries derived from gilts at age of 30–400 days were fixed in 10% (w/v) paraformaldehyde with 0.02 MPBS (pH 7.2) at 4°C for about 2 h. Next, they were cut into vertical slices of about 0.5 cm thickness and fixed in fresh 10% (w/v) paraformaldehyde for a cumulative period of 24 h. These slices were mounted in paraffin, and serially cut into 5 μm-thick sections by Rotary Microtome (MICROM, Germany), and stained with HE. Ovarian HE sections were observed and photographed under a fluorescent microscope (Zeiss, Germany).
Immunohistochemistry detection was performed by using the anti-Rabbit HRP-DAB Cell and tissue staining kit (R&D, CTS005) and anti-Goat HRP-DAB Cell and tissue staining kit (R&D, CTS008). Immunohistofluorescence examination was performed by using TSA plus Fluorescein (PerkinElmer, NEL741001KT) and Cyanine3.5 (PerkinElmer, NEL763001KT) kit. Antibodies of BMP15 (Eterlife, EL806306-100), GDF9 (Eterlife, EL901942-100), FSHR (Eterlife, EL912710), luteinizing hormone receptor (LHR) (Eterlife, EL904141), Caspase3 (Abcam, ab13847), 3βHSD (Abcam, ab154385), p-Smad1/5 (CST, 9516), Ki67 (Abcam, ab15580) and LC3B (Arigo, ARG55799) were diluted 1:100 with PBS. Other antibodies including ALK6 (Santa Cruz, sc5679), BMPR2 (Santa Cruz, sc5683), Smad2/3 (Santa Cruz, sc8332), Smad1/5/8 (Santa Cruz, sc6031R), and p-Smad2/3 (Santa Cruz, sc11769) were diluted 1:50 in PBS.

For immunohistochemical staining, 4 µm kidney sections were prepared. For antigen retrieval, the sections were heated at 80 °C for 30 min in 10 mmol/L citrate buffer solution at pH 6.0. Endogenous peroxidase blocking was conducted in 3.0% H₂O₂ in methanol for 20 min. Further blocking was conducted in normal goat serum at room temperature for 20 min. The sections were then incubated overnight at 4 °C with mouse polyclonal anti-VSIG4 (1:200; R&D system, Minneapolis, MS, USA). The sections were stained using the anti-goat HRP-DAB cell and tissue staining kit (R&D system) and counterstained with Mayer’s hematoxylin. Negative tissue controls were prepared under identical conditions with buffer solution and without primary antibodies. For VSIG4 immunostaining, four scores were semi-quantitatively assigned based on the extent of positive glomerular cells and tubulointerstitial fields: 0, absent or <25% positive area; 1, 25–50% positive area; 2, 50–75% positive area; and 3, >75% positive area.