Dec 20, 2023
Immunohistochemistry data processing
- Tae-Un Han1
- 1National Institute of Health
Protocol Citation: Tae-Un Han 2023. Immunohistochemistry data processing. protocols.io https://dx.doi.org/10.17504/protocols.io.x54v9p5omg3e/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: December 20, 2023
Last Modified: June 01, 2024
Protocol Integer ID: 92522
Keywords: ASAPCRN, high throughput quantification of immunohistochemistry data, immunohistochemistry data, immunohistochemistry, mouse brain, high throughput quantification, data
Funders Acknowledgements:
ASAP
Grant ID: ASAP-000458
Abstract
This protocol has been used for high throughput quantification of immunohistochemistry data of mouse brain sections
Materials
1. Zeiss AxioScan.Z1 slide scanning microscope system (Carl Zeiss Inc., Thornwood, NY, USA) with a Plan-Apochromat 20x/0.8 objective lens.
2. Hamamatsu Orca flash 4.0 camera
3. The Zeiss ZEN blue 2.3 software
4. MediaCybernetics’ Image-Pro 11 software package (Rockville, MD, USA).
Troubleshooting
Widefield fluorescent images are collected for GFP and DAPI fluorescent channels using a Zeiss AxioScan.Z1 slide scanning microscope system with a Plan-Apochromat 20x/0.8 objective lens.
All images are acquired using a Hamamatsu Orca flash 4.0 camera with an average tile count of 165 tiles per brain section.
The Zeiss ZEN blue 2.3 software package was used for collection and stitching of the 2-color (DAPI & GFP) tiled images.
Widefield fluorescent images are then post-processed using MediaCybernetics’ Image-Pro 11 software package (Rockville, MD, USA).
Every stitched image is processed using a protocol modified for each antibody. Initially, the image is masked to solely include the tissue in areas of interest.
Threshold segmentation is used for each antibody staining to separate actual signal from background auto-fluorescence.
Smart Segmentation is used to separate GFP-expressing puncta or fibrils from DAPI-stained nuclei in tissue evaluated with each antibody.
The Count/Size function is designed to extract the percent of the tissue sample that stains positive for GFP.