This protocol is significant because it allows for quantification of two proteins in the same brain region. This allows the experimenter to look at activity in molecular signaling pathways in brain regions by assaying pan and phosphoproteins. It can also be used to assay proteins that are markers of different cognitive phenomena.
We are using a relatively simple technique, such as immunohistochemistry, to answer questions that typically require more involved techniques. Including examining the activity of molecular signaling pathways, examining AMPA/NMDA ratio. Using a cryostat maintained between minus 9 degrees Celsius and minus 12 degrees Celsius, slice previously isolated and frozen rat brain in regions of interest at 30-50 micrometers.
Directly mount slices onto glass slides and store at minus 80 degrees Celsius until immunocytochemistry. Remove the glass slides from the freezer and allow them to equilibrate to room temperature for 30 minutes. Working under a fume hood, place the slides in 4%paraformaldehyde in 0.1 molar PBS for one to two hours at room temperature for tissue fixation.
Then rinse the slides in 0.1 molar TBS three times for 10 minutes each. To permeabilize cell membranes, incubate the slides in a mild detergent for 30-60 minutes. And rinse again in TBS three times for 15 minutes each.
Dilute primary antibody for protein of interest in PBS in the correct concentration as described in the manuscript. Pipette primary antibody solution directly onto the brain tissue. Place cover slips on the slides and incubate the brain tissue in primary antibody dilution for 1-2 hours at room temperature.
After incubation, remove the cover slips and wash the slides in TBS that has a small amount of detergent added to it, four times for 15 minutes each. Dilute secondary antibody in a diluent containing TBS, detergent, and 1.5%of host serum. Add secondary antibody to the slides, cover with cover slips and incubate at room temperature for two hours.
After incubation, rinse the slides in TBS-T four times for 20 minutes each and then in TBS four times for 20 minutes each. Dry the slides at room temperature in the dark, overnight. Place slides onto near-infrared scanning interface with the tissue facing down.
Image multiple slides at a time using the selection tool. Image the slides using the highest quality setting with a resolution of 21 micrometers. In an offset of zero nanometers, import images into image analysis software to view and mark for semiquantitative protein analysis.
After opening the image analysis software, select the work area into which the image was scanned. Then open the scanned image in the image analysis software to view the scan and adjust wavelengths, contrast, brightness, and magnification shown without altering the raw image or the total quantified emission. After identifying the key regions for quantification, select the analysis tab along the top of the page, then select draw rectangle to draw a rectangle over the area that will be quantified.
To view the rectangle size, select shapes along the bottom left of the screen. Then select columns along the bottom right. Then add height and width columns to identify the shape size.
Finally, name the shape and repeat. Once all regions are sampled, proceed with combining and analyzing of the data available from the columns tab. To verify that this protocol works, brain sections were incubated with primary and secondary antibodies, secondary antibody alone, or neither primary nor secondary antibody.
The immediate early gene c-jun expression was detected in the dorsal hippocampus and amygdala only when both primary and secondary antibodies were applied to the brain tissue. GluR1 sub-unit of AMPA receptor and the NR2A sub-unit of NMDA receptor were assayed in the stool protein detection in amygdala nuclei. This allowed examining the ratio of AMPA NMDA receptors in sub-nuclei of the amygdala which is a neurobiological signature of learning and memory.
Mean and normalized measures of protein expression were obtained from high resolution scans in the ventral hippocampus. Using the image analysis software, a rectangle is placed in the region of interest and mean intensity of light from the shape was used as a measure of fluorophore expression, which is a measure of protein expression. To obtain the normalized curve, a shape was placed across a region that expresses high signal and low signal in the area under the curve was used as a measure of protein expression.
The biggest struggle with this procedure is finding an antibody and making sure that it works. The application is simple. My advice would be to first attempt a regular immunohistochemical procedure, then try this technique.
Always performing a validation assay first. The most important thing to remember is to check your antibody dilution and make sure it's applied correctly. Without these steps, the procedure will fail.
