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.

When ready to begin, immunostaining, allow the sections to come up to room temperature. Then use a paintbrush to transfer every fifth section from a series of hippocampal sections to 25-well staining nets placed in Petri dishes filled with PBS. There should be 8 to 12 sections per hippocampus for staining. Transfer the staining nets, containing the sections, to matching glass dishes, containing PBS, and wash the sections twice for 10 minutes in PBS.

Then, incubate in 3% hydrogen peroxide for 20 minutes. After washing, move the sections through three hydrochloric acid solutions. Then, neutralize in 0.1 molar sodium tetraborate at room temperature for 20 minutes.

After three 10-minute incubations in changes of washing buffer, transfer the sections to blocking solution for one hour at room temperature. Then, incubate the sections in mouse anti-BrdU, diluted 1 to 100 in blocking solution for 48 hours at 4 degrees Celsius.

After the primary antibody incubation, wash three times for 10 minutes each in washing buffer, then incubate for 48 hours in horseradish peroxidase, diluted 1 to 10 in washing buffer. 48 hours later, transfer the sections to PBS for 10 minutes, and repeat for a total of five washes. After washing, transfer the sections to DAB solution for seven minutes. Then, transfer to DAB solution containing 0.02% hydrogen peroxide for 10 minutes.

After a series of washes, mount the sections in numerical order on microscope slides. Allow to dry for approximately 30 minutes. Then, air dry the samples by placing them into a slide rack. Next, rehydrate the samples for 10 minutes in a slide staining dish containing distilled water.

Then, transfer the slides to 0.02% crystal violet in distilled water for 15 minutes. After repeating the crystal violet stain, rehydrate the sections through an ethanol series. Then, clear the sections in xylene twice for 15 minutes each time. Finally, add rapid drying mounting medium, and coverslip the slides.

After 24 hours, the slides can be used for microscopy. Begin by checking the thickness of the tissue by first placing the slides on the motorized stage of the microscope, and then opening the stereology software. Delineate the area of interest using a low magnification objective before changing to a 100x oil immersion objective.

Then, pick a specific point in accounting frame, for example, an area adjacent to a corner, and locate the top of the section by moving along the focal plane until some feature of the section appears in focus. Register this z position as 0. Next, move the focal plane down through the tissue until the last z level of tissue is in focus, then mark this position. The local tissue thickness is defined from 0 to this endpoint, and can be read on the z-axis.

Register the tissue thickness. The tissue thickness is measured in several places within the region of interest. The height of the dissector and the guard zones, the size of the counting, frame the step length, and the final magnification is determined in a pilot study. Now, you can begin to count the BrdU-positive neurons, usually at a final magnification of 2,000 to 3,000x, using a 60x oil, or 100x oil objective, and a high numerical aperture.

Identify BrdU-positive neurons in each counting frame, but do not count those that touch the red exclusion line. Count those BrdU-positive neurons that touch the green inclusion line, and those that fall within the boundary of the counting frame. Only count BrdU-positive neurons when the feature of interest is clearly recognized within the dissector height. The semi-visible neurons shown here are not counted.