The overall goal of this procedure is to perform antigen retrieval on hippocampal brain tissue to better visualize the progenitor cell types giving rise to newborn neurons. This is accomplished by first harvesting the perfused brain from mouse cutting thin coronal sections, and mounting these sections on a glass slide, specifically designed to adhere strongly to the tissue. The second step is to boil the slides with tissue and Kay atopic treatment and to allow sufficient time for cooling.
Next, stain the tissue overnight with primary antibodies. The final step is to perform secondary antibody staining on the tissue the next day for at least two hours. Ultimately, visualize the various pro gender cell types across the various stages of development during adult neurogenesis of the hippocampal tissue with time lapse or confocal microscopy.
Hi, my name is Hun Jo, a postdoc, a member of Dr.Ian John's lab and Mayo Clinic. Our main focus in John Lab is investigating the process of other neurogenesis in neurological and psychiatric disorders. Our end goal is to successfully translate such knowledge into therapeutic application demonstrating our procedure of antigen retriever will be Quam Hui, and hi John from our laboratory.
The main advantage of our technique over existing methods of immunohistochemistry is that it allows effective visualization of distinct cell types during adult hippocampal neurogenesis. These cell types, including the precursor neural stem cells that are otherwise difficult to detect, can be efficiently labeled and visualized using our method of antigen retrieval. This method will provide insight into the development and regulation of newborn neurons during adult hippocampal neurogenesis.
For those interested in the study of mental disorders and pharmacological treatment, our antigen retrieval will prove useful in investigating the role of neuronal development during this disease.Pathogenesis. Begin the procedure by placing the brain from a perfused mouse in 4%para formaldehyde in a 10 milliliter conical tube overnight at four degrees Celsius, or for at least 12 to 24 hours, but no more than 36 hours for full fixation. After overnight fixation, remove PFA and leave the brain in the conical tube.
Next, rinse the brain with 30%sucrose to remove any residual PFA and discard the sucrose. Then fill the tube with 30%sucrose again and keep it at four degrees Celsius until the brain sinks to the bottom of the tube. Afterwards, cut the hippocampus into 40 micrometer brain sections on a microtome.
Preserve the sections in the antifreeze solution until they are ready for immunohistochemistry. Then mount the slices on the microscope slides that are specifically designed to adhere to the tissue strongly. After mounting the tissue, let the slides dry for about 10 minutes.
Drying can be expedited if the slides are rested against a vertical surface on a paper towel to ensure complete removal of previous chemicals. Once the slides are dry, wash them with PB S3 times for five minutes each time. Then let it dry again.
In this procedure, prepare the solution for tissue boiling according to the description in the accompanying manuscript. Next, boil the solution for five minutes in a standard microwave at standard setting. Once boiling is complete, carefully remove container from microwave.
After that, transfer the dried slides into the solution. Make sure that the side with the hippocampal sections is fully exposed to the solution. If possible, place the slides at an angle against the walls of the container and stack other slides around them.
Then boil the mixture with the slides for seven minutes in the microwave at standard settings. In the meantime, fill two ice buckets halfway with ice. Once the boiling is complete, remove the container from the microwave and place it inside one of the ice buckets.
Pour the rest of the ice from the other bucket all around the container and completely cover it. After that, let it sit for one hour after an hour. Remove the slides from the container.
Wash them with TBST buffer three times for five minutes each time. Next, dry the slides in dark for five to 10 minutes or until slide is fully dry. In the meantime, prepare a chamber with DDH two O for overnight primary antibody staining.
Once the slides have dried, draw an outline around the tissue sections using a water appellant pen. This outline acts as a barrier to prevent the antibody mixture from flowing over. Then prepare the primary antibody mixture in T-B-S-T-T.
Place the dried slides on the stage in the staining chamber. Add 500 microliters of the primary antibody slowly to cover the entire surface. Subsequently, cover the staining chamber to block external light and keep it overnight at room temperature on the next day, wash the slides three times for five minutes each time with TBST buffer.
After that, dry them in the dark. Now prepare the secondary antibody mixture in T-B-S-T-T. Then place the dry slides in the staining chamber.
Add 500 microliters of the secondary antibody. Slowly cover the staining chamber and keep it from external light for at least two hours at room temperature after the completion of secondary staining, wash the slides three times for five minutes each time with TBST buffer, dry the slides in dark after drying. Add mounting solution to the slides.
Cover them with cover slips carefully in order to prevent the introduction of any bubbles. Then keep the slides in dark for at least two hours before subsequent analysis and performing time lapse or convo analysis on the stained tissue. Adult hippocampal neurogenesis is depicted across the sub granular zone of the dentate gyrus.
An activated radial glial like type one stem cell passes through distinct phases as it differentiates, matures and functionally integrates with an elaborate morphology into the neuronal circuit. Each of the phases and the respective cell types can be identified based on their expression of a combination of markers. MCM two is a proliferation marker that is expressed across all the mitotic progenitor cell types while nein is expressed among radial and non radial glial like stem cells.
As the type two A progenitors differentiate nested expression is first turned off. Anti TBR two expression increases in the post mitotic progenitor MCM two expression is lost while DCX and NEWAN expression dictate migration and maturation into a functional neuron. This is a representative confocal image of purple MCM two green neston and red dappy staining.
The magnified images in the boxes show a radial GL like progenitor expressing neston MCM two and dpi. Here is a representative confocal image of blue MCM two red, TBR two green DCX, and gray dpi. Staining the characteristic of a Type two B progenitor cell type is shown in the magnified images in the boxes expressing MCM two, TBR 2D, c, X, and dpi.
While attempting this procedure, it's important to remember three things. One, utilize well perfused and fixed tissue of the right coronal thickness. Two, mounting the tissue flat on the slides and preventing any damage while doing so.
And finally, three, allowing sufficient time for boiling and subsequent cooling during antigen retrieval. Following this procedure, other methods like confocal tissue reconstruction and behavior experiments can be performed in order to answer additional questions in regards to how newborn neurons may integrate themselves into the hippocampal circuitry and contribute to cognitive function. After watching this video, you should have a good understanding of how to perform antigen retriever on your hippocampal tissue to visualize with greater effectiveness, the presentative cell types that give a rise to new neurons during other hippocampal neurosis.
