May 14, 2024

Public workspaceQuantification of IHC Using an Inverted Confocal Microscope and NIS-Elements Program-Killinger Lab 2024

DOI
https://dx.doi.org/10.17504/protocols.io.5qpvokrbzl4o/v1
  • Solji Choi1
  • 1Rush University Medical Center
Bryan Killinger
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DOI: https://dx.doi.org/10.17504/protocols.io.5qpvokrbzl4o/v1
Protocol Citation: Solji Choi 2024. Quantification of IHC Using an Inverted Confocal Microscope and NIS-Elements Program-Killinger Lab 2024. protocols.io https://dx.doi.org/10.17504/protocols.io.5qpvokrbzl4o/v1
Manuscript citation:
Choi SG, Tittle T, Garcia-Prada D, Kordower JH, Melki R, Killinger BA. Alpha-synuclein aggregates are phosphatase resistant. bioRxiv [Preprint]. 2024 Apr 9:2023.11.20.567854. doi: 10.1101/2023.11.20.567854. PMID: 38645137; PMCID: PMC11030248.
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: May 14, 2024
Last Modified: May 14, 2024
Protocol Integer ID: 99764
Keywords: quantification of ihc, ihc, using ni, stained section, killinger lab, inverted confocal microscope, confocal microscope, dab, killinger lab 2024 protocol
Funders Acknowledgements:
Michael J. Fox Foundation
Grant ID: ASAP-021030
MIchael J. Fox Foundation
Grant ID: ASAP-024442
Abstract
Protocol to quantify DAB stained sections using NIS-Elements.
Capture brightfield images with an inverted confocal microscope with a 20X objective (Nikon A1R).
Conduct annotation of each tissue section within a bounding box of 2000×2000 pixels for mouse tissues and 2863×2454 pixels for human tissues.
Use a manual RGB-based color thresholding algorithm for mouse tissues to exclude pure methyl green nuclei staining from the measurement.  For human tissues, use NIS-elements (version 5.10.01, https://www.microscope.healthcare.nikon.com/products/software/nis-elements, RRID:SCR_014329) auto-thresholding algorithm.
Export the percentage area of the thresholded signal (object area fraction provided in the program) and normalize to the average value of non-CIAP treated tissues.