Goat anti rabbit ig hrp

Sections were dewaxed, rehydrated, and subjected to heat-induced antigen retrieval by incubating in 0.1 M citrate buffer (pH 6.0, Abcam, catalog ab93678). Endogenous peroxidase was blocked with 3% H₂O₂ (MilliporeSigma, catalog H1009) in TBS (pH 7.4) for 30 minutes. Sections were then blocked with 10% normal goat serum in 5% BSA at room temperature for 2 hours. Phospho-S6 ribosomal protein (Ser240/244) rabbit mAb (Cell Signaling Technology, catalog 5364) was used at a concentration of 1:1,000 in a cold room (4°C) overnight. Antibody binding was detected by using secondary antibody, goat anti-rabbit Ig-HRP (1:100, Southern Biotech, catalog 1010-05), and the Betazoid DAB Chromogen Kit (Biocare Medical, catalog BDB2004). Counterstaining was performed using hematoxylin. Two pathologists who were blinded to the participants’ clinical data scored the intensity of the phospho-S6 staining as follows: 0 indicated less than 5% of cells stained, 1 indicated from 5% to 25% of cells stained, 2 indicated from 25% to 50% of cells stained, and 3 indicated more than 50% of cells stained. Separate scores were created for the proximal and distal nephron segments, and the scores of the 2 pathologists were averaged.