Apr 23, 2026
Immunocytochemistry with mDANs
Forked from Immunocytochemistry for TH-positive neurons
- Ellen Hertz1,
- Yu Chen1,
- Ellen Sidransky1
- 1National Institutes of Health
Protocol Citation: Ellen Hertz, Yu Chen, Ellen Sidransky 2026. Immunocytochemistry with mDANs. protocols.io https://dx.doi.org/10.17504/protocols.io.q26g7n619lwz/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: October 09, 2025
Last Modified: April 23, 2026
Protocol Integer ID: 229431
Keywords: ASAPCRN, positive neurons immunocytochemistry protocol for ipsc, positive neurons immunocytochemistry protocol, derived dopaminergic neuron, dopaminergic neuron, immunocytochemistry for th, immunocytochemistry, ipsc, immunocytochemistry with mdans immunocytochemistry protocol, mdans immunocytochemistry protocol, mdan
Funders Acknowledgements:
ASAP
Grant ID: ASAP-000458
Abstract
Immunocytochemistry protocol for iPSC-derived dopaminergic neurons
Protocol materials
NucBlue™ Fixed Cell ReadyProbes™ ReagentThermo FisherCatalog #R37606
Geltrex™ LDEV-Free, hESC-Qualified, Reduced Growth Factor Basement Membrane MatrixThermo FisherCatalog #A1413302
Paraformaldehyde solution 4% in PBSSanta Cruz BiotechnologyCatalog #sc-281692
GFP tag Polyclonal antibodyProteintechCatalog #50430-2-AP
Mitofusin-1 (D6E2S) Rabbit mAbCell Signaling TechnologyCatalog #14739
StrepMAB-ClassicIBA LifesciencesCatalog #2-1507-001
Sheep Tyrosine HydroxylasePel-FreezCatalog #P60101-150
RFP Antibody Pre-adsorbedROCKLAND antibodes & assaysCatalog # 600-401-379
HNF-3β/FoxA2 antibodyNeuromicsCatalog #GT15186
Mouse anti-Human GCase antibody (Clone 1/23)Michael J Fox Foundation (MJFF) Research Tools Catalog
LAMP1 (D2D11) XP® Rabbit mAb (PE Conjugate)Cell Signaling TechnologyCatalog #85995
mDAN Seeding in Imaging Chambers
Coat 8-well imaging chambers (Ibidi Cat# 80807-90) with Geltrex 37 °C for 01:00:00 .
Geltrex™ LDEV-Free, hESC-Qualified, Reduced Growth Factor Basement Membrane MatrixThermo FisherCatalog #A1413302
Dissociate Day 25 mDANs with Accutase for 00:15:00
Resuspend dissociated mDANs in Neurobasal media + B27 w/o vitamin A + CEPT
Seed mDANs in Geltrex-coated imaging chamber at 50~100k cells/ cm2.
Immunocytochemistry
On day 35; fix mDANs inParaformaldehyde solution 4% in PBSSanta Cruz BiotechnologyCatalog #sc-281692 for 00:30:00 atRoom temperature
Wash cells 00:05:00 in 200 µL DPBS with gentle rocking. Repeat for a total of 3 washes
Add blocking solution (1% BSA, 0.1% saponin in PBS) for 1 hour at Room temperature
Dilute primary antibodies in blocking buffer
Sheep Tyrosine HydroxylasePel-FreezCatalog #P60101-150 1:400
RFP Antibody Pre-adsorbedROCKLAND antibodes & assaysCatalog # 600-401-379 1:500
HNF-3β/FoxA2 antibodyNeuromicsCatalog #GT15186 1:500
Mouse anti-Human GCase antibody (Clone 1/23)Michael J Fox Foundation (MJFF) Research Tools Catalog 1:1000
LAMP1 (D2D11) XP® Rabbit mAb (PE Conjugate)Cell Signaling TechnologyCatalog #85995 1:500
GFP tag Polyclonal antibodyProteintechCatalog #50430-2-AP 1:1000
Mitofusin-1 (D6E2S) Rabbit mAbCell Signaling TechnologyCatalog #14739 1:100
StrepMAB-ClassicIBA LifesciencesCatalog #2-1507-001 1:100
Incubate with primary antibodies at 4 °C Overnight
Wash cells 5 minutes in 200 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 Room temperature
Wash cells 5 minutes in 200 µL PBS with gentle rocking. In second wash include NucBlue™ Fixed Cell ReadyProbes™ ReagentThermo FisherCatalog #R37606
Image cells in DPBS