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Immunohistochemical (IHC) staining

YH Yating Huang
ZM Zhijie Ma
XK Xiaojun Kuang
QZ Qiuwan Zhang
HL Haiyan Li
DL Dongmei Lai
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After deparaffinization and hydration, the ovarian sections were placed in sodium citrate solution and boiled for antigen retrieval. The following process was performed using an IHC kit (Abcam). Briefly, sections were treated with hydrogen peroxide and blocked, followed by incubation with primary antibodies against von Willebrand factor (vWF, newly formed vessels marker, 1:500, Boster), CD34 (endothelial marker, 1:2000, Abcam), and DDX4 (oocyte marker, 1:2000, Abcam). Then, the sections were washed and sequentially incubated with biotinylated anti-mouse/rabbit IgG, streptavidin peroxidase, and DAB chromogen solution, and finally, counterstained with hematoxylin. The negative control samples received identical treatment, except primary antibodies were omitted, and exhibited no specific staining. For quantification, four ovarian sections from each mouse were subjected to IHC, and three randomly selected high-power fields in each section were analyzed. The final data represent the average microvessel density (MVD).

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