The overall goal of this stereological study using the optical fractionator is to obtain quantitative information, such as the total number of cells in a specific brain area, using methods based on unbiased principles. This method can help answer key questions in the field of quantitative analysis, such as estimation of the total number of cells in a complete structure of interest. One main benefit of this technique is that it provides accurate estimates with a fixed and pre-determined precision.
Demonstrating this procedure is Esther Kjaer Needham, who is a co-author of the article. Begin with a fixed and frozen rat brain. Using a scalpel, separate the right and left hemispheres along the midline of the brain and then choose one or the other hemisphere randomly in the first brain.
Thereafter shift systematically between right and left hemisphere. Then use mounting medium to mount the chose hemisphere onto a specimen disc with a long axis perpendicular to the disc. When the hemisphere is mounted on the disc place a tube around the hemisphere and fill it with Tissue-Tek to completely cover the hemisphere.
Next, place numbered multi dish containers into the cryostat for pre-cooling. Then mount the specimen disc into the cryostat and cut the entirety of the hippocampus starting randomly into 80 micron thick sections in the coronal plane. While cutting, place all sampled sections consecutively in the cold multi dish containers to ensure that the sections are kept in cutting order.
When the whole hippocampus has been sectioned cover the sections completely with cryoprotectant and keep the containers at minus 20 degree Celsius until further use. When ready to begin immunostaining allow the sections to come up to room temperature. Then use a paint brush 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 eight 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 to three hydrochloric acid solutions. Then neutralize in 0.1 sodium tetraborate at room temperature for 20 minutes. After three 10-minute incubations and 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 one to 100 in blocking solution for 48 hours at four degree 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 one 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%cresyl violet and distilled water for 15 minutes. After repeating the cresyl 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 cover slip 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 a counting 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 the Z position as zero. 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 zero 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 Brd-U positive neurons, usually at a final magnification of 2000 to 3000x 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 Brd-U positive neurons when the feature of interest is clearly recognized within the dissector height. The semi-visible neurons shown here are not counted. This image shows the total number of BrdU-positive neurons in the hippocampal granule cell layer and subgranular zone of the rat hippocampus.
The horizontal bars represent the mean values. Following electroconvulsive stimulation there is an immediate 260%increase in the formation of new BrdU-positive neurons compared to when treated controls. In this pool of newly generated neurons there is 40%attrition from day one to three months with nearly 50%of the newly formed neurons surviving 12 months following treatment.
After watching this video you should have a good understanding of how to get an estimation of the total number of cells in a brain region using stereological methods.
