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Methoxychlor-Induced Atresia: MATERIALS AND METHODS(10)

METHODS(10)

To ensure that proteins were loaded equally in each lane, the blots were stripped using ImmunoPure elution buffer (Pierce) and incubated with a p-actin (1:3000 dilution, Santa Cruz Biotechnology) and HRP-conjugated anti-mouse polyclonal antibody. Scanning densitometry was used to estimate the pixel density of individual bands using Molecular Analyst Software (Bio-Rad Laboratories, Hercules, CA).

RNA Isolation and Real-Time Polymerase Chain Reaction Analysis

To compare FSH receptor (FSHR) gene expression between MXC-treated and sesame oil-treated ovaries, we used real time-PCR. Briefly, ovaries were collected from sesame oil- and MXC-treated animals, snap frozen, and stored at —70°C until further processing. Total RNA was extracted from whole ovaries using the RNeasy Mini Kit (Qiagen, Inc., Valencia, CA) according to the manufacturer’s protocol. Real-time quantitative PCR was conducted using a MJ Research (OPTICON) Real-Time PCR machine (MJ Research, Waltham, MA) and accompanying software according to the manufacturer’s instructions. The OPTICON quantifies the amount of PCR product generated by measuring the dye (SYBR green) that fluoresces when bound to double-stranded DNA.