The overall goal of this immunohistochemical staining method is to visualize combined expression of axon sorting molecules at the axon termini of olfactory sensory neurons. This method can help characterize key processes in the field of olfactory development, such as axon guidance, synapse formations, and the regeneration of olfactory sensory neurons. The main advantage of this technique is that it allows scientists to compare and analyze the expression patterns of axon sorting molecules in olfactory bulb without sustaining variation between sections.
Visual demonstration of this method is critical as the embedding step is difficult to learn, because the position and angle of the olfactory tissues sample in the mold are hard to describe in words. Start with heads from mice of one to two weeks of age that have been fixed by intracardiac perfusion of 4%paraformaldehyde. Insert a scissor blade into the space between the upper and lower teeth, and cut horizontally to remove the lower jawbone.
Then trim away excess tissue with forceps and scissors, to leave the olfactory tissue, including the olfactory bulb and the olfactory epithelium. Immerse the olfactory tissue in PBS to remove air in the nasal cavity, then ake a vertical cut to remove the posterior part of the brain, and place the olfactory tissue on the bottom of an embedding mold with the cut end face down. Fill the mold with optimal cutting temperature compound, and then immerse in liquid nitrogen.
After the tissue and OCT is completely frozen, equilibrate the tissue in the cryostat at 20 degrees celsius for one hour. Now make serial parasaggital sections of the olfactory bulb with the cryostat, and collect them by sticking to MAS coated glass slides. After sticking, dry the slides immediately with a blow dryer.
Begin immunostaining by washing the dried slides with PBS for five minutes at room temperature. Block nonspecific binding sites by incubating the slides for one hour with 5%blocking solution at room temperature. Then add 400 microliters of a cocktail of primary antibodies diluted in 1%blocking solution to each slide.
Incubate the slides overnight at room temperature. The next day, discard the primary antibody solution and wash the slides with PBST three times for five minutes each at room temperature. Then add 400 microliters of a cocktail of secondary antibodies and PBS to each slide.
Protect from light and incubate for one hour at room temperature. Following the incubation, discard the solution and again wash the slides three times for five minutes each with PBS at room temperature. Finally, mount cover slips on the slides by applying two drops of mounting medium to each slide, placing the cover slip, and removing air bubbles.
Obtain fluorescent images with the fluorescence microscope using the appropriate filter for each fluorophor. Adjust the exposure time to obtain fluorescent signals of the image without saturation. Define the glomerular structure by immunofluorescent signals of GLUT2.
Use image J to measure staining intensities of axon sorting molecules within glomeruli. This image shows a parasaggital olfactory bulb section from a two week old mouse immunostained with antibodies against VGLUT2, Kirrel2 shown in red, Semaphorin 7A shown in green, and OLPC in blue. The merged image demonstrates that the axon sorting molecules involved in glomerular segregation are expressed in a position-independent mosaic pattern.
Each glomerulus is defined by fluorescent signals of the presynaptic marker VGLUT2. The glomerular structures are surrounded by dashed lines. Signal intensities of axon-sorting molecules were measured in each glomerulus.
As seen here, axon sorting molecules are differentially expressed in each glomerulus. For example, glomerulus 3 has high levels of OLPC, intermediate levels of Semaphorin 7A, and lower levels of Kirrel2, and this difference can be quantified by measuring the staining intensity. Whereas glomerulus 4 has high levels of OLPC, and lower levels of Kirrel2 and Semaphorin 7A.
Again, this difference can be quantified by measuring the staining intensity. While attempting this procedure, it's important to remember to omit post-fixation with PFA, and treatment with sucrose solution, which are normally recommended. After watching this video, you should have a good understanding of how to place more glomeruli on the single olfactory bulb section, and how to perform high quality immunostaining with multiple antibodies, which can be applied to other brain areas.
Don't forget to hope, this method will help you succeed in your future experiments.
