Sep 17, 2020

Public workspaceImmunofluorescence and Cell Counting

This protocol is a draft, published without a DOI.
  • Yingchao Xue1,2,
  • Xiping Zhan3,
  • Shisheng Sun4,
  • Senthilkumar S. Karuppagounder5,6,7,
  • Shuli Xia2,5,
  • Valina L Dawson5,6,7,8,9,
  • Ted M Dawson5,6,7,8,10,
  • John Laterra2,5,8,11,
  • Jianmin Zhang1,
  • Mingyao Ying2,5
  • 1Department of Immunology, Research Center on Pediatric Development and Diseases, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences and School of Basic Medicine, Peking Union Medical College, State Key Laboratory of Medical Molecular Biology;
  • 2Hugo W. Moser Research Institute at Kennedy Krieger;
  • 3Department of Physiology and Biophysics, Howard University;
  • 4College of Life Sciences, Northwest University;
  • 5Department of Neurology, Johns Hopkins University School of Medicine;
  • 6Neuroregeneration and Stem Cell Programs, Institute for Cell Engineering, Johns Hopkins University School of Medicine;
  • 7Adrienne Helis Malvin Medical Research Foundation;
  • 8Department of Neuroscience, Johns Hopkins University School of Medicine;
  • 9Department of Physiology, Johns Hopkins University School of Medicine;
  • 10Department of Pharmacology and Molecular Sciences, Johns Hopkins University School of Medicine;
  • 11Department of Oncology, Johns Hopkins University School of Medicine
  • Neurodegeneration Method Development Community
    Tech. support email: ndcn-help@chanzuckerberg.com
Anita Broellochs
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External link: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6344911/
Protocol Citation: Yingchao Xue, Xiping Zhan, Shisheng Sun, Senthilkumar S. Karuppagounder, Shuli Xia, Valina L Dawson, Ted M Dawson, John Laterra, Jianmin Zhang, Mingyao Ying 2020. Immunofluorescence and Cell Counting. protocols.io https://protocols.io/view/immunofluorescence-and-cell-counting-9vah62e
Manuscript citation:
Synthetic mRNAs Drive Highly Efficient iPS Cell Differentiation to Dopaminergic Neurons. Xue Y, Zhan X, Sun S, Karuppagounder SS, Xia S, Dawson VL, Dawson TM, Laterra J, Zhang J, Ying M. Stem Cells Transl Med. 2019 Feb;8(2):112-123. doi: 10.1002/sctm.18-0036. Epub 2018 Nov 1. PMID: 30387318
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
These protocols were published in: Xue Y, Zhan X, Sun S, et al. Synthetic mRNAs Drive Highly Efficient iPS Cell Differentiation to Dopaminergic Neurons. Stem Cells Transl Med. 2019;8(2):112–123. doi:10.1002/sctm.18-0036
Created: November 28, 2019
Last Modified: September 17, 2020
Protocol Integer ID: 30338
Keywords: ND1014, N1, ND27760, ipsc, SNCA, Atoh2, Ngn2, immunofluorescence
Abstract
This protocol explains Immunofluorescence and Cell Counting for lines ND1014, N1, and ND27760 from Synthetic mRNAs Drive Highly Efficient iPS Cell Differentiation to Dopaminergic Neurons.
Guidelines
Antibodies for immunofluorescence staining
Antibody Species Company Catalog # Dilution factor
MONOCLONAL ANTI-FLAG(R) M2-HRP Mouse Sigma-Aldrich A8592-.2MG 1000
LMX1A Rabbit millipore AB10533 100
FOXA2 Rabbit CST 8186P 200
FOXA2 Mouse R&D Systems AF2400-SP 100
DAT Rat millipore MAB369 200
TH Rabbit CST 2792S 200
Nurr1 Rabbit millipore PA14519 200
β3-Tubulin (Tuj1) Mouse Covance MMS-435P 1000
β3-Tubulin (Tuj1) Rabbit CST 5568 1000
Synapsin Rabbit CST 2312 200
GIRK2 Rabbit Abcam ab66502 200
Myosin IIa Rabbit CST 3403S 1000
Myosin IIb Rabbit CST 3404S 1000
Neurogenin 2 Rabbit CST 13144S 1000
Materials
MATERIALS
ReagentTriton(R) X-100 100mlPromegaCatalog #H5142
Reagent4% Paraformaldehyde in PBSAlfa AesarCatalog #J61899-AK
ReagentGoat SerumGibco - Thermo Fisher ScientificCatalog #16210-064
ReagentDAPIThermo Fisher ScientificCatalog #62248
ReagentProLong™ Gold Antifade MountantThermo FisherCatalog #P36930
Safety warnings
Please refer to the Safety Data Sheets (SDS) for safety and environmental hazards.
Before start
Obtain approval to work with human stem cells from an appropriate Institutional Review Board.
Immunofluorescence
Immunofluorescence
Fix cells in 4% paraformaldehyde in PBS Ph7.4 .
Block cells in 5% normal goat serum and 0.2% Triton X-100.
Dilute primary antibodies in 5% normal goat serum. Primary antibodies are listed in the "Guidelines".
Incubate samples with primary antibodies DurationOvernight at Temperature4 °C .

Incubation
Stain using Cy3‐ and Alexa Fluor 488‐labeled secondary antibodies.
Counterstain with DAPI.
Mount on glass slides using ProLong antifade.
Pause
Cell Counting
Cell Counting
Randomly select 10 different fields to use for counting the number of DAPI-positive cells expressing specific markers in ImageJ. Perform this with at least three independent experiments.