This is a key protocol in the Adult Neurogenesis field. Here we present a detailed description of the steps required to achieve a successful staining. This technique makes it possible to simultaneously identify specific proteins co-expressed in the cells under division while preserving tissue integrity.
We recommend testing the tissue antibodies and conditions in advance. The crucial steps are membrane permeabilization, careful DNA denaturation, and testing the times for blocking and washing steps. Standardizing the technique is challenging for new laboratories.
The reagent preparation and administration of thymidine analog in rodent are critical to successful outcomes For thymidine analog BrdU administration, immobilize the lower abdominal cavity to restrain the experimental rat and use a one milliliter syringe equipped with a 23 gauge needle to intraperitoneally inject a freshly prepared 50 milligram per kilogram BrdU solution into the animal. At the appropriate experimental endpoint, collect the brain and freeze the brain to allow the acquisition of 40 micrometer thick coronal sections using a cryostat microtome and place the sections into individual wells of a 24 well cell culture plate in cryo protection solution in the order of their collection. When all of the sections have been collected, store the samples at minus 20 degrees Celsius for up to a few months.
To assess BrdU staining, using a peroxidase reaction, with the avidin-biotin-peroxidase complex, transfer the slices from the cryo protection solution to 0.1 molar PBS until the samples reach room temperature and rinse the tissues three times for 10 minutes with fresh 0.1 molar PBS per wash. After the last wash, incubates the sections in endogenous peroxidase blocking solution for 30 minutes, at room temperature, followed by three 10 minute washes in 0.1 molar PBS. After the last wash incubate the sections for 20 minutes at 37 degrees Celsius in two molar hydrochloric acid followed by one 10 minute rinse in 0.1 molar borate buffer.
After three washes in ice cold 0.1 molar PBS incubate the samples for two hours at room temperature in PBS+blocking solution, followed by an overnight incubation with mouse Anti-BrdU primary antibody in PBS+at four degrees Celsius. After three 0.1 molar PBS washes, incubate the samples for two to four hours with the appropriate mouse biotinylated secondary antibody in PBS+at room temperature. After washing, treat the sections with ABC Solution for one hour at room temperature, followed by three 0.1 molar PBS washes.
After the last wash transfer slices to DAB-Peroxidase Substrate Solution for a two to 10 minute incubation until the slices become dark gray. If positive cells can be visualized, rinse the samples with three 15 minute tap water washes followed by three 0.1 molar PBS washes. After the last wash Use a soft brush to carefully mount the slices on gelatinized slides, for overnight air drying at room temperature.
The next morning use permanent mounting medium to place cover slips over the samples. To quantify the BrdU positive cells, first, use the 4x magnification, on a microscope, to identify the dentate gyrus and carefully search the granular cell layer along the Z axis for BrdU labeled nuclei. After selecting an interval section for cell searching all over the dentate gyrus, count all BrdU positive cells.
The morphology of the labeled nucleus can change depending on how much BrdU the cell incorporated. Move slowly over the Z axis to quantify all nuclei from at least 10 sections per animal and at least five animals per group that integrate a cluster. For image deconvolution, open the diffraction point spread function 3D plugin, fill out all the required data, click OK and save the file.
Next, open the DeconvolutionLab2 plugin and drag the matched Z-stack image and PSF file to the corresponding window slot. Select the deconvolution algorithm, and the number of iterations, and click run. To combine the deconvoluted images into a single Z-stack image, select Image and Stacks and click Z project.
From the projection type menu, select Max Intensity. Click OK and save the file. To create an RGB image, select Image and Color and click Merge Channels to set the corresponding image to the desired color channel from the dropdown menu.
Then uncheck the Create composite box. Click OK and save the file. When all of the channel images have been created open at least two of the image files and select different color channels for each image file to create an RBG image.
In these images, representative dentate gyrus sections with BrdU labeled cells can be observed. As observed in these magnified images of the sections the labeled cells demonstrate an intense dark staining. Off note, there are significant differences in BrdU positive cell numbers between controlled rats that did not undergo physical activity and the experimental group that was exposed to physical activity, consistent with other reported results that have demonstrated that physical activity increases cellular proliferation in the adult dentate gyrus.
When attempting this protocol employ freshly prepared solutions and handle the tissue gently. The hippocampus is susceptible to separate from the rest of the slides when treated through it and can get missed. This procedure could be adapted for DNA label and index evaluation of DNA synthesis and doubling staining cell proliferation studies.
We hope that this effort has been helpful to the scientific community and makes it easier to answer new questions in the study of cell proliferation, when immunohistochemistry technique.
