Measuring Protein Expression in the Rodent Brain Using Near-Infrared Fluorescence and High-Resolution Scanning

Take a glass slide containing rat brain tissue sections immunostained for AMPA and NMDA receptors in amygdala neurons.

AMPA receptors mediate glutamate-induced sodium influx and fast excitatory synaptic transmission. 

In contrast, NMDA receptors mediate glutamate and co-agonist-induced sodium and calcium influx, facilitating slow excitatory synaptic transmission.

The receptors are stained with primary antibodies and detected using near-infrared fluorophore-tagged secondary antibodies.

Place the slide tissue-side down on the near-infrared scanning interface.

Adjust the imaging parameters and begin imaging.

Upon laser illumination, the fluorophores on the AMPA and NMDA receptors emit fluorescence.

Near-infrared imaging minimizes autofluorescence, enhancing signal clarity.

High-resolution scanning further ensures clear differentiation of fluorescence signals, enabling precise visualization of nearby AMPA and NMDA receptors in the amygdala.

Using imaging software, calculate the AMPA/NMDA fluorescence intensity ratio in a specific region of interest as a marker of learning and memory.

Place slides onto near-infrared scanning interface with the tissue facing down. Image multiple slides at a time using a selection tool. Image the slides using the highest quality setting, with a resolution of 21 micrometers. In an offset of 0 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.