Oct 24, 2025

Immunostaining  V.3

DOI
https://dx.doi.org/10.17504/protocols.io.n92ld62d8g5b/v3
  • 1Weill Cornell medicine;
  • 2Weill Cornell Medicine;
  • 3Aligning Science Across Parkinsons;
  • 4Collaborative Research Network
Jacquelyn Haytayan
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DOI: https://dx.doi.org/10.17504/protocols.io.n92ld62d8g5b/v3
Protocol Citation: Sabina Marciano, Jacquelyn Haytayan, Roberta Marongiu 2025. Immunostaining . protocols.io https://dx.doi.org/10.17504/protocols.io.n92ld62d8g5b/v3Version created by Jacquelyn Haytayan
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: October 24, 2025
Last Modified: October 24, 2025
Protocol  Integer ID: 230699
Keywords: ASAPCRN, immunostaining , Histology , psyn , Antibody, microglial marker iba1, synuclein aggregate, multiplexed fluorescent detection of dopaminergic neuron, synuclein, microglia, neurodegenerative features in parkinsonian model, study of neuroinflammatory, neurodegenerative feature, neuroinflammatory, pathological markers in aof, dopaminergic neuron, endogenous peroxidase, pathological marker, multiplexed fluorescent detection, pd tissue, immunohistochemistry protocol, parkinsonian model, quenching of endogenous peroxidase, phosphorylated alpha
Funders Acknowledgements:
Aligning Science Across Parkinsons
Collaborative Research Network
Abstract
This immunohistochemistry protocol was developed to visualize pathological markers in AOF-PD tissue, focusing on phosphorylated alpha-synuclein (psyn), tyrosine hydroxylase (TH), and microglial marker Iba1, 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
Primary Antibodies:
  • Goat anti-Iba1 Iba1, WAKO, cat. 01919741 1:1500
  • Rabbit anti-TH Abcam, cat. Ab113,  1:1000
  • Mouse anti-Myc Cell Signaling, cat. 2276
  • Mouse anti-psyn Abcam cat. ab51253 1:1000

Secondary Antibodies:
  • Donkey anti-goat 594 1:1000
  • Donkey anti-rabbit 647 1:500
  • Donkey anti-mouse 488 1:1000
  • Neurotrace 455 (1:100)

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
Washes Wash 3x in PBS-T, 10min each (0.2% Triton in PBS)
Quench Incubate in 3% H2O2 in PBST for 2hrs at room temperature in the DARK! (x3)
Can be done the day before and sections can be stored in PBS overnight at 4 degrees C
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.
Goat anti-Iba1 Iba1, WAKO, cat. 01919741 1:1500
Rabbit anti-TH Abcam, cat. Ab113, 1:1000
Mouse anti-Myc Cell Signaling, cat. 2276
Mouse anti-psyn Abcam cat. ab51253 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-goat 594 1:1000
Donkey anti-rabbit 647 1:500
Donkey anti-mouse 488 1:1000
Neurotrace 455 (1:100)
1) Washes Wash 3x in PBS-T, 10min each