Apr 13, 2026
Version 1
Immunoperoxidase Histology and Densitometric Quantification for Tyrosine Hydroxylase V.1
- Roberta Marongiu1,
- Sabina Marciano2
- 1Weill Cornell Medical College;
- 2Weill Cornell Medicine
Protocol Citation: Roberta Marongiu, Sabina Marciano 2026. Immunoperoxidase Histology and Densitometric Quantification for Tyrosine Hydroxylase. protocols.io https://dx.doi.org/10.17504/protocols.io.36wgqxkkylk5/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 13, 2026
Last Modified: April 13, 2026
Protocol Integer ID: 314942
Keywords: ASAPCRN, densitometric quantification for tyrosine hydroxylase, standardized workflow for tyrosine hydroxylase, cultivation of rat dopaminergic n27 cell, reproducible densitometric quantification in the striatum, tyrosine hydroxylase, rat dopaminergic n27 cell, automated quantification of immunofluorescent marker, immunoperoxidase, immunofluorescent marker, reproducible densitometric quantification, histology
Funders Acknowledgements:
Aligning Science Across Parkinson's
Collaboration Research Network
Abstract
This protocol describes the cultivation of rat dopaminergic N27 cells, transient transfection and immunoblottingThis protocol details the systematic acquisition and automated quantification of immunofluorescent markers (TH, mCherry, Iba1, and pSer129) in the Substantia Nigra (SN) and striatum. It utilizes a blinded workflow involving QuPath for regional delineation and CellProfiler for object and intensity quantification.This protocol describes a standardized workflow for Tyrosine Hydroxylase (TH) immunoperoxidase staining using the ABC-DAB method. Sections were processed as one batch so that reagent exposure is kept identical, ensuring highly reproducible densitometric quantification in the striatum and Substantia Nigra (SN).
Tissue Preparation and Pooling
Select one SN and one striatal section per animal.
Punch code the cortex of the sections to allow for identification after pooling.
Pool tissue sections from all experimental groups into a single container to ensure identical reagent exposure.
Immunoperoxidase Staining (Day 1-3)
Rinse: Wash sections in 0.1M PB followed by 0.1M TS (90 rpm).
Block: Incubate in 0.5% BSA in TS for 30 min (90 rpm).
Primary Antibody: Incubate in sheep anti-TH (1:5000) in 0.1% Triton-X/0.1% BSA/TS.
24 hours at Room Temperature (145 rpm).
24 hours at 4 degrees (145 rpm).
Secondary Antibody: Rinse in TS, then incubate in biotin-conjugated donkey anti-sheep IgG (1:400) in 0.1% BSA/TS.
ABC Incubation: Wash in TS and incubate in ABC complex (at 50% of the manufacturer’s recommended dilution) for 30 min.
Visualization and Mounting
DAB Reaction: Visualize bound peroxidase using DAB and 0.003% H2O2 in TS for 10 minutes.
Mounting: Mount sections in 0.05 M PB onto gelatin-coated glass slides.
Dehydration: Run slides through an ascending alcohol series (e.g., 70%, 95%, 100%).
Clearing & Coverslipping: Clear in Xylene and coverslip using DPX.
Image Acquisition
Use the Nikon Eclipse 80i and Micropublisher 5.0 camera to capture images.
Utilize IP Lab software for consistent digital acquisition.
Ensure the investigator performing acquisition is blinded to the experimental conditions.
Densitometric Quantification
Open images in ImageJ64.
Define Regions of Interest (ROI) in the striatal regions.
Background Subtraction: Measure pixel density in a non-labeled region (e.g., anterior commissure).
Subtract this background value from the ROI density to control for illumination variability.
Convert pixel density to transmittance values for final analysis.