Rats were anesthetized (5 mg/100 g, intraperitoneal injection) and euthanized by cervical dislocation on day 35, and bilateral ovaries were collected for subsequent experiments. Ovarian tissues were treated with RIPA lysis buffer (Wuhan Boster Biological Technology, Ltd., Wuhan, PRC). The protein concentration of each sample was determined with a BCA protein quantification kit (Wuhan Boster Biological Technology, Ltd.). Membranes were blocked with 5% dried skimmed milk (Wuhan Boster Biological Technology, Ltd.). The antibodies used in the experiment mainly include rabbit polyclonal anti-NGF (1 : 1000), rabbit polyclonal anti-FSHR (1 : 500), rabbit polyclonal anti-TrkA (1 : 500), and rabbit polyclonal anti-p75 (1 : 500; all Abcam, Cambridge, UK), goat anti-rabbit IgG secondary antibody (1 : 5000, Thermo Fisher Scientific, Inc.). The protein contents in the sample were calibrated and quantified with GAPDH (1 : 1000, Abcam) as internal reference. Immunodetection was performed using SuperSignal™ West Dura Extended Duration substrate (Thermo Fisher Scientific, Inc., Waltham, MA, USA). The X-ray films were developed, images were captured, and Quantity One software (Bio-Rad, Hercules, CA, USA) was used to analyze the gray values. The specific method of this experiment is shown in our previous research [13 (link)].
Free full text: Click here
