Parvalbumin Immunostaining

To effectively section the brains, we cryogenically froze and preserved them. After sectioning, we identified tissue sections around the target region based on hippocampal development from the hundreds of slices we made. We also selected tissues anterior and posterior to the target region, which we identified again based on hippocampal development, which was greater in posterior regions and less defined in anterior regions. When it came time to stain and plate the tissues, we repeatedly rinsed them in phosphate-buffered saline (PBS) buffer in order to remove any residue from the freezing, in which the brain was glued down.

The sections were treated with bovine serum albumin and donkey serum albumin and subsequently donkey anti-mouse secondary antibody to avoid cross reaction with the primary antibody, mouse anti-parvalbumin, which the tissues were kept in overnight. Treatment with mouse peroxidase anti-peroxidase followed, then staining with diaminobenzidine (DAB), hydrogen peroxide (which oxidizes DAB), and nickel ammonium sulfate.

The DAB staining gives treated cells a brown color, and the nickel ammonium sulfate enhances its effect to give a black stain. The DAB stain will be used for the subsequent cholinergic immunostaining, which is an antibody-based method to detect a specific protein in a sample, as well as the current parvalbumin immunostaining. The DAB and nickel staining provides a cost effective alternative to fluorescent staining, since we don’t have a fluorescent microscope. Luckily, the tissue sections stained, which was not a given, as if any step was  The slides were rinsed in a gelatinizing solution we prepared, and the treated target region, anterior, and posterior tissues from each of the three brains were plated.They were then rinsed in solutions of 70%, 95%, and 100% alcohol, followed by xylene before placing cover slips on them.