The overall goal of this procedure is to describe a simple technique to optimize the staining and visualization of estrogen receptor alpha using cross-sectional slices of peel arterials obtained from female rat brains. This is accomplished by first removing Evan's blue dye stained peel arterials from the brain's surface and cutting them into eight micron rings using a cryostat after sectioning. The cross sections are mounted on slides and incubated with the primary antibody against estrogen receptor alpha.
Next, the sections are incubated with the secondary antibody and counterstain using mounting media containing dpi. Finally, the fluorescently labeled peel vessel segments are imaged and converted into 2D images using extended depth focus software. Ultimately, results can be obtained that show peel arteriolar estrogen receptor alpha through the use of a Nikon immunofluorescent microscope with extended depth software.
My name is Dr.Marguerite Littleton Kearney. Today, Dr.Neil Far ani, a postdoctoral fellow in my laboratory will demonstrate a simple procedure in order to optimize the visualization and staining of estrogen receptor alpha. In peel.
Arterials Thor a rat brain that has been perfused with Evan's blue stain and stored at minus 70 degrees Celsius. Using a dissecting microscope, identify the peel arteries on the surface of the brain and remove them using fine tipped forceps. Carefully removing any a adherent brain tissue.
Place the arteries in a small test tube on ice. Fix the arteries in 2%cold power formaldehyde in 0.1 mole of PBS for 30 minutes. To prepare the arteries for sectioning pour embedding medium on a freezing disc set at minus 20 degrees Celsius.
Place a section of artery flat on the chug and wait for it to freeze. Once it has frozen firmly, place the artery upright in embedding media on the cryostat freezing disc. Ensure that the tip of the artery is facing forward.
Hold the artery with tweezers until it is solidly frozen. Place the freezing disc with the artery into the cryostat head and then cut eight micron peel arterial rings directly onto a slide. Mount the arterial sections on chromium potassium gelatin.
Sub slides using two paintbrushes to straighten the arterial rings. Store the prepared slides in a slide box in the refrigerator at four degrees Celsius overnight. Remove the slides from the refrigerator and bring them to room temperature on the bench top.
Wash the slides with one to 1.5 milliliters of 0.1 molar PBS After 10 minutes, drain the PBS and repeat twice more. After the final PBS wash, incubate the slides in one milliliter of 50 millimolar ammonium chloride for 30 minutes at room temperature In order to reduce endogenous fluorescence, wash the slides in 0.1 molar PB S3 times at 10 minutes per wash as previously described. Next, block the slides in a PBS solution containing 0.1%Triton X 100 plus 1%normal goat serum for 30 minutes to reduce non-specific binding of the primary antibody.
After blocking, incubate the slides with the primary antibody against ER alpha diluted to a concentration of one to 500 in the blocking solution overnight at four degrees Celsius. Remove the slides from the refrigerator and bring them up to room temperature. Wash off any unbound primary antibody with one to 1.5 milliliters of 0.1%PB S3 times for 10 minutes.
Each time, incubate the slides with Oregon Green, 4 88 secondary anti rabbit in blocking solution for two hours in the dark at room temperature after incubation, wash the slides in 0.1 molar PB S3 times at 10 minutes per wash, and then bring them to a ventilated hood. Place a drop of mounting media containing dappy on the vessels, and then place a cover slip over the sample. Apply clear nail polish to seal the edges of the cover slip and allow it to dry completely.
Insert the prepared slides with the stained peel vessel segments under the microscope and adjust to visualize the fluorescently labeled areas of interest. Visualization of the stained ER alpha is the most difficult step of this procedure. Find the level of the cut vessel with the clearest receptors.
Start at 100 times magnification and then increase the magnification to 600 times. Use the camera to capture images in DPI and ZI channels fo cell nuclei and DR alpha respectively, and then use software to convert multiple views of vessel segments into 2D images.Here. The results of immunofluorescent staining for ER alpha are shown in green and counter staining with DPI.
In blue in a peel artery ring isolated from a female rat brain, the merged images suggest the presence of intranuclear estrogen receptor alpha. By using a combination of Zack images, a focused image of the ER alpha immunofluorescence can be achieved to account ver autofluorescence negative controls for both the primary and secondary antibody should be performed. Note the distinct autofluorescence along the endothelial side of an arterial that has not been incubated with secondary antibody.
After watching this video, you should have an appreciation of how to isolate, peel arterials from the brain surface and to prepare eight micron cross-sectional rings. Additionally, you should be familiar with how fluorescently labeled estrogen receptor alpha, and to visualize the receptors using a digital immunofluorescent microscope and Nikon extended that software.
