Goat anti rabbit immunoglobulin g igg secondary antibody

Djulis was obtained from Sinfong Agritech Co. (Taipei, Taiwan). Iron (III) chloride hexahydrate, methylene blue, and acetic acid were purchased from Nacalai Tesque (Tokyo, Japan), Showa Chemicals (Tokyo, Japan), and Shimakyu Pure Chemicals (Osaka, Japan), respectively. The Bax primary antibody and Bcl-2 primary antibody were purchased from Cell Signaling Technology (Beverly, MA, USA). Glyceraldehyde-3-phosphate dehydrogenase and the caspase-9 primary antibody were purchased from GeneTex (Irvine, CA, USA). The proliferating cell nuclear antigen (PCNA) primary antibody, goat anti-rabbit immunoglobulin G (IgG) secondary antibody, and peroxidase AffiniPure goat anti-mouse IgG were purchased from Abcam (Cambridge, UK), Southern Biotechnology (Birmingham, AL, USA), and Jackson ImmunoResearch (West Grove, PA, USA), respectively. The β-actin primary antibody, 1,2-dimethylhydrazine (DMH), N,N’-dimethyl-p-phenylenediamine, N,N’-dimethyl-m-phenylenediamine, Alcian blue, and the remaining chemicals were purchased from Sigma Chemical (St. Louis, MO, USA).

Granulosa cells were revealed by positive staining of the follicle-stimulating hormone receptor (FSHR). Granulosa cells were treated with a rabbit anti-FSHR primary antibody (cat# 3929, Sigma-Aldrich, Saint Louis, MO, USA) and then a goat anti-rabbit immunoglobulin G (IgG) secondary antibody (cat# ab6717, Abcam, Cambridge, UK).
For the analysis of mitochondrial membrane potential, granulosa cells were concurrently treated with N, N, N’, N’-tetra-methylethylenediamine (TMRE) (cat# T669, Molecular probes, Life Technologies, Auckland, NZ) or MitoTracker Red FM (cat# M22425, Molecular probes) at 37 °C for 60 min, washed with PBS and centrifuged at 250 g for 15 min at room temperature to remove excess dye. For the analysis of mitochondrial mass, granulosa cells were stained with nonyl acridine orange (NAO) (cat# A1372 Molecular probes) at 37 °C for 30 min. Following staining and washing with PBS, the samples were centrifuged at 250 g for 15 min at room temperature to remove excess dye.
Flow cytometry analysis was performed using a FACSCalibur system (Becton, Dickinson and Company, BD Biosciences; USA) with the side-scattered light (SSC) detector voltage set at 415, the FL1 detector voltage set at 635, the FL2 detector voltage set at 614, and the FL3 detector voltage set at 150 voltage.