Jan 06, 2026
Immunostaining
- Roberta Marongiu1,
- Sabina Marciano2
- 1Weill Cornell Medical College;
- 2weill cornell medicine
Protocol Citation: Roberta Marongiu, Sabina Marciano 2026. Immunostaining . protocols.io https://dx.doi.org/10.17504/protocols.io.14egn1z16v5d/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: January 06, 2026
Last Modified: January 06, 2026
Protocol Integer ID: 237151
Keywords: ASAPCRN, immunofluorescence, endogenous peroxidase activity, primary antibodies against tyrosine hydroxylase, fluorescent secondary antibody, tyrosine hydroxylase, procedure with hydrogen peroxide quenching, staining procedure, detection of th, hydrogen peroxide quenching, primary antibody, reduced autofluorescence, secondary antibody, bsa before overnight incubation, fluorescent
Funders Acknowledgements:
Aligning Science Across Parkinson's
Collaborative Research Network
Weill Cornell Medicine
Abstract
This protocol describes an immunofluorescence staining procedure with hydrogen peroxide quenching to reduce endogenous peroxidase activity, optimized for detection of TH and Myc/Cherry signals. Tissue sections are permeabilized with PBS-T, quenched with 3% H₂O₂ in the dark, and blocked with BSA before overnight incubation with primary antibodies against tyrosine hydroxylase (TH) and Myc. Fluorescent secondary antibodies and NeuroTrace are then applied for visualization, with thorough PBS-T washes between steps to minimize background. The protocol supports robust, specific labeling with reduced autofluorescence and background.
Materials
Solutions:
PBS-T Triton 0.2 % 150mL: 300uL Triton in 150mL PBS
Before start
All washing steps are performed for 10min and on a shaker
Immunostaining: AOF-PD: myc/cherry, TH quench
Washes Wash 3x in PBS-T, 10min each (0.2% Triton in PBS)
Quench Incubate in 3% H2O2 in PBST for 2hrs at RT in the DARK! (x3) (can be done the day before and sections can be stored in PBS ON at 4degrees)
Washes Wash 3x in PBS-T, 10min each (0.2% Triton in PBS)
Blocking Incubate in 3% BSA/PBS-T for 1 hr at room
temperature.
Primary Antibody Incubate in primary antibody (diluted in blocking solution) overnight.
Rabbit anti-TH Abcam, cat. Ab113, 1:1000
Mouse anti-Myc Cell Signaling, cat. 2276 1:1000
Washes Wash 3x in PBS-T, 10min each
Secondary Antibody Incubate in secondary antibody (diluted in blocking solution) 1hr at room temperature.
Donkey anti-rabbit 647 1:500
Donkey anti-mouse 488 1:1000
Neurotrace 455 (1:200)
Washes Wash 3x in PBS-T, 10min each