Mar 29, 2024
Optical Density Analysis for DAB Staining of Mouse Brain Sections
- Katerina Rademacher1,
- Ken Nakamura1
- 1Gladstone Institute of Neurological Disease
Protocol Citation: Katerina Rademacher, Ken Nakamura 2024. Optical Density Analysis for DAB Staining of Mouse Brain Sections. protocols.io https://dx.doi.org/10.17504/protocols.io.81wgbxo2nlpk/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: January 07, 2024
Last Modified: May 31, 2024
Protocol Integer ID: 93033
Keywords: ASAPCRN
Funders Acknowledgement:
ASAP
Grant ID: 020529
Abstract
This protocol describes steps for optical density analysis of immunohistochemical 3,3′-Diaminobenzidine (DAB) staining of mouse brain sections using ImageJ.
Materials
- FIJI/ImageJ Software (RRID:SCR_002285)
Open ImageJ, then calibrate for analysis.
See https://imagej.net/ij/docs/examples/calibration/ for calibration setup steps and files.
Select Analyze > Calibrate, then select the following:
i. Rodbard (NIH Image)
ii. Unit: O.D.
iii. Open the calibration.txt file
iv. Uncheck ‘show plot’
v. Check ‘Global calibration’
Open an image and outline the regions of interest, including a background area for subtraction.
Select ‘multi measure’ and copy the results to an Excel sheet for analysis. Repeat for all images.
Take the ‘mean OD’ value for each region and subtract the background area ‘mean OD’ value. Values can be plotted directly or normalized to control groups.