Anti-Choline Acetyltransferase Antibody AB5042

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"Immunohistochemical staining was performed using a three-stage streptavidin-peroxidase method following the instructions of the test kit. Briefly, 5 μm tissue sections were heated at 60°C followed by de-waxing treatment and permeabilization. Antigen retrieval was performed using citrate salt buffer for 25 min, followed by incubation with 3% H2O2 for 10 min at room temperature. After washing three times with PBS, goat serum was added for 20 min to block nonspecific antigen binding.

The rabbit anti-NF-κB and rabbit anti-ChAT primary antibodies were added and incubated overnight at 4°C. After washing in PBS, the tissue sections were incubated for 30 min in horseradish peroxidase (HRP)-conjugated rabbit anti-mouse IgG secondary antibody. Tissue sections were washed three times in PBS, for 5 min each time, and the chromogenic substrate was added, followed by rinsing with tap water. Hematoxylin counterstaining was followed by rinsing with 0.1% HCl-ethanol, followed by dehydration in graded ethanols and mounting in resin. Counting of positive cells was performed by light microscopy under ×100 objective. Four separate fields were selected within the hippocampal CA3 region followed by analysis of the integrated optical density (IOD) by IPP version 6.0 software. The average value was calculated for the number of positive cells."

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