Quantifying Newly Generated Neurons in Rat Brain Tissue Using the Optical Fractionator Technique

Take hippocampal brain slices from a rat injected with Bromodeoxyuridine, or BrdU, a nucleoside analog of thymidine.

BrdU integrates into DNA during cell division, labeling newly generated neurons within the dentate gyrus, a region for neurogenesis. 

Incubate with an oxidizing agent to inactivate endogenous peroxidase enzymes.

Treat with acid to denature DNA and expose BrdU, then neutralize the acid.

Incubate with a detergent to permeabilize cellular membranes and a blocking solution to mask non-specific binding sites.

Incubate with primary antibodies targeting BrdU, followed by peroxidase-tagged secondary antibodies specific to the primary antibodies.

Introduce a chromogenic substrate and an oxidizing agent, allowing peroxidase to convert the substrate into a colored residue.

Mount the tissue and counterstain with a nucleic acid-binding dye.

Dehydrate the tissue, then treat it to increase transparency.

Under a microscope, identify the dentate gyrus.

Using the optical fractionator technique, analyze multiple planes across the tissue thickness to quantify BrdU-labeled neurons.