Sep 23, 2025

Public workspaceImmunocytochemistry for TH-positive neurons

DOI
https://dx.doi.org/10.17504/protocols.io.8epv5k38jv1b/v1
  • Ellen Hertz1,
  • Yu Chen1,
  • Ellen Sidransky1
  • 1National Institutes of Health
Ellen Hertz
Icon indicating open access to content
QR code linking to this content
DOI: https://dx.doi.org/10.17504/protocols.io.8epv5k38jv1b/v1
Protocol Citation: Ellen Hertz, Yu Chen, Ellen Sidransky 2025. Immunocytochemistry for TH-positive neurons. protocols.io https://dx.doi.org/10.17504/protocols.io.8epv5k38jv1b/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: September 23, 2025
Last Modified: September 23, 2025
Protocol Integer ID: 228005
Keywords: ASAPCRN, positive neurons immunocytochemistry protocol for ipsc, positive neurons immunocytochemistry protocol, derived dopaminergic neuron, dopaminergic neuron, immunocytochemistry for th, immunocytochemistry, ipsc
Funders Acknowledgements:
ASAP
Grant ID: ASAP-000458
Abstract
Immunocytochemistry protocol for iPSC-derived dopaminergic neurons
Protocol materials
ReagentGeneticin™ Selective Antibiotic (G418 Sulfate) (50 mg/mL)Thermo FisherCatalog #10131019
ReagentParaformaldehyde solution 4% in PBSSanta Cruz BiotechnologyCatalog #sc-281692
ReagentGeltrex™ LDEV-Free, hESC-Qualified, Reduced Growth Factor Basement Membrane MatrixThermo FisherCatalog #A1413302
ReagentNucBlue™ Fixed Cell ReadyProbes™ ReagentThermo FisherCatalog #R37606
ReagentSheep Tyrosine HydroxylasePel-FreezCatalog #P60101-150
ReagentAnti-Neomycin Phosphotransferase II AntibodyMerck MilliporeSigma (Sigma-Aldrich)Catalog #06-747
Troubleshooting
Cell culture
Seed cell in GeltrexReagentGeltrex™ LDEV-Free, hESC-Qualified, Reduced Growth Factor Basement Membrane MatrixThermo FisherCatalog #A1413302 coated 8-well imaging chambers (Ibidi Cat# 80807-90) at differentiation day 25 in media according to Kim, T.W., et al., Biphasic Activation of WNT Signaling Facilitates the Derivation of Midbrain Dopamine Neurons from hESCs for Translational Use. Cell Stem Cell, 2021. 28(2): p. 343-355 e5 differentiation scheme.

Om DIV 27 change media to media including geneticin ReagentGeneticin™ Selective Antibiotic (G418 Sulfate) (50 mg/mL)Thermo FisherCatalog #10131019

Immunocytochemistry
On day 37; fix cells inReagentParaformaldehyde solution 4% in PBSSanta Cruz BiotechnologyCatalog #sc-281692 for 15 minutes atTemperatureRoom temperature

Wash cells 5 minutes in Amount200 µL PBS with gentle rocking. Repeat for a total of 3 washes

Add blocking solution (1% BSA, 0.1% saponin in PBS) for 1 hour at TemperatureRoom temperature

Dilute primary antibodies e.g. ReagentSheep Tyrosine HydroxylasePel-FreezCatalog #P60101-150 1:400 and/or ReagentAnti-Neomycin Phosphotransferase II AntibodyMerck MilliporeSigma (Sigma-Aldrich)Catalog #06-747 1:500 in blocking buffer and incubate at Temperature4 °C DurationOvernight

Wash cells 5 minutes in Amount200 µL PBS with gentle rocking. Repeat for a total of 3 washes
Dilute secondary antibodies from Jackson ImmunoResearches matching primary antibodies in blocking buffer and incubate 1 hour at TemperatureRoom temperature

Wash cells 5 minutes in Amount200 µL PBS with gentle rocking. In second wash include ReagentNucBlue™ Fixed Cell ReadyProbes™ ReagentThermo FisherCatalog #R37606

Image cells in PBS