Quantification of IHC Using an Inverted Confocal Microscope and NIS-Elements Program-Killinger Lab 2024
Solji Choi
Published: 2024-05-14 DOI: 10.17504/protocols.io.5qpvokrbzl4o/v1
Abstract
Protocol to quantify DAB stained sections using NIS-Elements.
Steps
1.
Capture brightfield images with an inverted
confocal microscope with a 20X objective (Nikon A1R).
2.
Conduct annotation of each tissue section within a bounding box of 2000×2000 pixels for
mouse tissues and 2863×2454 pixels for human tissues.
3.
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.
4.
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.
