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.j8nlk141wg5r/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: 237149
Keywords: ASAPCRN, IHC, Immunostaining , Parkinson's, Gene Therapy , microglial marker iba1, multiplexed fluorescent detection of dopaminergic neuron, synuclein aggregate, microglia, study of neuroinflammatory, neurodegenerative features in parkinsonian model, neurodegenerative feature, neuroinflammatory, synuclein, pathological markers in aof, immunohistochemistry, pd tissue, multiplexed fluorescent detection, endogenous peroxidase, dopaminergic neuron, immunohistochemistry protocol, pathological marker, tyrosine hydroxylase
Funders Acknowledgements:
Aligning Science Across Parkinson's
Collaborative Research Network
Weill Cornell Medicine
Abstract
This immunohistochemistry protocol was developed to visualize pathological markers in AOF-PD tissue, focusing on tyrosine hydroxylase (TH), microglial marker Iba1, GFAP, and DAT with quenching of endogenous peroxidases to reduce background. The protocol enables multiplexed fluorescent detection of dopaminergic neurons, microglia, and pathological alpha-synuclein aggregates, facilitating the study of neuroinflammatory and neurodegenerative features in Parkinsonian models.
Materials
Solutions:
PBS-T Triton 0.2 % 150mL: 300uL Triton in 150mL PBS
Immunostaining: AOF-PD: TH, Iba1, GFAP, DAT
1) 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)
All washing steps are performed for 10min and on a shaker
Blocking Incubate in 3% BSA/PBS-T for 1 hr at room temperature.
5) Primary Antibody Incubate in primary antibody (diluted in blocking solution) overnight.
Goat anti-Iba1 Iba1, WAKO, cat. 01919741 1:1500
Rabbit anti-TH Abcam, cat. Ab113, 1:1000
Mouse anti-GFAP ThermoFisher cat. PA1-10019 1:1500
Rat anti-DAT Millipore Cat #MAB369 1:500
Washes Wash 3x in PBS-T, 10min each
All washing steps are performed for 10min and on a shaker
Secondary Antibody Incubate in secondary antibody (diluted in blocking solution) 1hr at room temperature.
Donkey anti-goat 594 1:1000
Donkey anti-rabbit 488 1:500
Donkey anti-mouse 405 1:1000
Donkey anti-rat 647 1:1000
Washes Wash 3x in PBS-T, 10min each
All washing steps are performed for 10min and on a shaker