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

Abstract

Source: Kimmelmann-Shultz, B., et. al. Using Near-infrared Fluorescence and High-resolution Scanning to Measure Protein Expression in the Rodent Brain. J. Vis. Exp. (2019)

This video demonstrates a near-infrared fluorescence and high-resolution scanning method for measuring protein expression in the rodent brain.

Protocol

All procedures involving animal samples have been reviewed and approved by the appropriate animal ethical review committee.

1. Imaging

1. Place slides onto the near-infrared scanning interface with the tissue facing down. Either image one glass slide or multiple slides at a time using a selection tool.

2. Image slides using the highest quality setting with an offset of 0 nm and a resolution of 21 μm. The scanning will typically take 13−19 h, depending on the scanning equipment used.

3. Import images into the image analysis software (e.g., Image Studio) to view and mark for semi-quantitative protein analysis.

2. Protein Expression Analysis

1. Open the image analysis software and select the Work Area into which the image was scanned.

2. Open the scanned image in the image analysis software to view the scan, and adjust which wavelengths are viewed, as well as the contrast, brightness, and magnification shown without altering the raw image or the total quantified emission.

3. Identify the key regions for quantification and select the Analysis tab along the top of the page, then select Draw Rectangle (or Draw Ellipse/ Draw Freehand) to draw a rectangle over the area that will be quantified.

4. To view the size of the rectangle, select Shapes along the bottom left of the screen, then select Columns along bottom right. Add Height and Width columns to identify the shape size.

NOTE: It is important to control for shape size when comparing quantification. It is recommended to use identical shapes placed within the desired quantification location, in order to get an accurate sampling of the emissions for that region.

5. Then name the shape and repeat. Once all regions are sampled the data available from the columns tab can be aggregated and analyzed.

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Glass Microscope Slides	Fischer Scientific	12-549-6
Licor Odyssey scanner	Licor Biotechnology Inc.
Image Studio	Licor Biotechnology Inc.