Mar 30, 2024
Optical Density Analysis for DAB Staining of Mouse Brain Sections
- Katerina Rademacher1,
- Ken Nakamura1
- 1Gladstone Institute of Neurological Disease
External link: https://doi.org/10.7554/eLife.98775
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
Manuscript citation:
Rademacher K, Doric Z, Haddad D, Mamaligas A, Liao S, Creed R, Kano K, Chatterton Z, Fu Y, Garcia JH, Vance VM, Sei YJ, Kreitzer A, Halliday G, Nelson AB, Margolis E, Nakamura K Chronic hyperactivation of midbrain dopamine neurons causes preferential dopamine neuron degeneration. eLife 13(). doi: 10.7554/eLife.98775
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: June 01, 2024
Protocol Integer ID: 93033
Keywords: ASAPCRN, optical density analysis for dab staining, staining of mouse brain section, optical density analysis, steps for optical density analysis, mouse brain section, dab staining, mouse brain sections this protocol, dab
Funders Acknowledgements:
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.