May 07, 2026
Semi-automated quantification of microglial CD68 and Iba1 immunohistochemistry signal in mouse brain slices
- Amandine Even1,2,3,4,5,
- Alex Tchung1,2,3,4,
- Nicolas Giguère1,2,3,4,
- Louis-Éric Trudeau1,6,2,3,4,5
- 1Department of pharmacology and physiology, Faculty of Medicine, Université de Montréal;
- 2Neural Signaling and Circuitry research group (SNC);
- 3Center for Interdisciplinary Research on the Brain and Learning (CIRCA);
- 4Institut Courtois d’innovation biomédicale;
- 5Aligning Science Across Parkinson’s (ASAP) Collaborative Research Network, Chevy Chase, MD, 20815;
- 6Department of neuroscience, Faculty of Medicine, Université de Montréal
Protocol Citation: Amandine Even, Alex Tchung, Nicolas Giguère, Louis-Éric Trudeau 2026. Semi-automated quantification of microglial CD68 and Iba1 immunohistochemistry signal in mouse brain slices. protocols.io https://dx.doi.org/10.17504/protocols.io.bp2l6j6zzvqe/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: April 21, 2026
Last Modified: May 07, 2026
Protocol Integer ID: 315472
Keywords: microglial cd68, iba1 immunohistochemistry signal in mouse brain slice, iba1 immunohistochemistry signal, floating brain section, quantification of cd68, mouse brain slice, brain section, iba1, cd68
Funders Acknowledgements:
Aligning Science Across Parkinson's
Grant ID: ASAP-000525
Abstract
Standardized procedure for semi-automated detection and quantification of CD68 and Iba1 in floating brain sections, using image acquisition, AI-based segmentation, and downstream data analysis.
Materials
- Slide scanner (Zeiss AxioScan 7)
- Analysis Workstation
- Zen software (Zeiss)
- NIS-Elements Advance Research software (Nikon) with GA3 pipelines Jobs (https://github.com/Louis-EricTrudeau/Trudeau-lab/tree/main/Mukherjee-2026/GA3), and Batch Deconvolution
- GraphPad Prism
Before start
A GA3 macro in NIS-Elements (Nikon imaging software) is an analysis script that automates image processing and quantitative measurements.
All GA3 macros necessary for this protocol are available here: https://github.com/Louis-EricTrudeau/Trudeau-lab/tree/main/Mukherjee-2026/GA3
Sample preparation
When sectionning the mouse brain in 40 µm coronal sections, collect one 40 µm coronal section out of every six for immunohistochemistry.
Perform immunohistochemistry labeling for DAPI, Iba1, CD68, and TH (tyrosine hydroxylase) using appropriate primary and secondary antibodies.
Image Acquisition on Zeiss AxioScan 7 slide scanner
Define regions of interest (ROI) per hemisphere from a pre-scan at 5x.
Three striatal coronal slices were selected per brain:
- The first slice was chosen at the level where the corpus callosum from both hemispheres was nearly touching.
- The second slice was selected at the level where the anterior commissure formed a straight line.
- The third slice included the hippocampus.
Capture reference screenshots of selected regions.
Acquire images using laser excitation of 405, 488, 546 and 647nm.
Scan sections to obtain 20 Z-stacks with Z-steps of 1µm, using a 40x objective.
Format Conversion and deconvolution
Convert images to TIFF format in batch using Zen software.
Processing - Batch
Batch Method: OME TIFF-Export
Method Parameters: see Figure 1
Figure 1
Convert images to ND2 file format for analysis in NIS software (Nikon).
If necessary, remove out-of-focus planes from each Z-stack.
Retain at least 15 planes per Z-stack and ensure that all images contain the same number of planes.
All the images were deconvolved with the Automatic function in BatchDeconvolution 6.10 NIS software.
Semi-automated quantification with NIS
Train the segmentation AI on NIS-Elements software to detect and count the microglia as described here.
Link the generated AI within the GA3.
Set all the tresholds and filters on multiple representative images.
Perform the automated quantification.
Data Analysis
Order the data from the csv file in Excel.
Import data into GraphPad Prism and generate plots.
6. Quality Control
Verify accuracy of each step visually after AI processing or batch macro analysis.