Aug 25, 2023
Immunofluorescence and live-cell Imaging
- Shenjie Wu1,
- Nancy C. Hernandez Villegas2,
- Iona Thomas-Wright3,
- Richard Wade-Martins3,
- schekman1
- 1Department of Molecular and Cell Biology, Howard Hughes Medical Institute, University of California, Berkeley, Berkeley, United States;
- 2Helen Wills Neuroscience Institute, University of California, Berkeley, Berkeley, United States;
- 3Oxford Parkinson’s Disease Centre, Department of Physiology, Anatomy and Genetics and Kavli Institute for Nanoscience Discovery, University of Oxford, Oxford, United Kingdom
Protocol Citation: Shenjie Wu, Nancy C. Hernandez Villegas, Iona Thomas-Wright, Richard Wade-Martins, schekman 2023. Immunofluorescence and live-cell Imaging. protocols.io https://dx.doi.org/10.17504/protocols.io.bp2l6x9b1lqe/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: August 03, 2023
Last Modified: August 25, 2023
Protocol Integer ID: 85900
Funders Acknowledgements:
Nancy C Hernandez Villegas
Grant ID: NIH training program T32GM139780
Randy Schekman
Grant ID: Howard Hughes Medical Institute
Abstract
This protocol contains a detail description of how to perform immunostaining on two different cell types, U2OS and iPSCs cells.
It also describes how to perform live-cell imaging procedure using a Zeiss LSM900 confocal microscope in a temperature-controlled environment.
Immunofluorescence of U2 cells
Immunofluorescence of U2 cells
50m
50m
U2OS cells were washed once with PBS and immediately fixed by 4% EM-grade paraformaldehyde for 00:10:00 at Room temperature
10m
Cells were washed three times with PBS for 00:10:00 each time.
10m
Blocked and permeabilized for 00:30:00 in permeabilization buffer (5% FBS and 0.1% saponin in PBS)
30m
Cells were then incubated with 1:100 dilution of primary antibodies at 4 °C Overnight
1h
Cells were washed three times with PBS for 00:10:00 each time.
10m
Cells were incubated with 1:500 dilution of fluorophore-conjugated secondary for 00:30:00 at Room temperature
30m
Prolong Gold with DAPI was used as mounting solution
Images were acquired with a Zeiss LSM900 confocal microscope and analyzed with Fiji/ImageJ software
Immunofluorescence of hiPSC dopamine neurons
Immunofluorescence of hiPSC dopamine neurons
2h 40m
2h 40m
Cells were fixed with 4% paraformaldehyde in PBS and 0.1% Triton-X was used for permablization 00:10:00
10m
Blocked in 10% normal donkey serum for 01:00:00 Room temperature
1h
Cells were then incubated with 1:100 dilution of primary antibodies at 4 °C Overnight
1h
Cells were washed three times with PBS for 00:10:00 each time.
10m
Cells were incubated with 1:500 dilution of fluorophore-conjugated secondary for 00:30:00 at Room temperature
30m
Prolong Gold with DAPI was used as mounting solution
Images were acquired with a Zeiss LSM900 confocal microscope and analyzed with Fiji/ImageJ software
Live-cell imaging
Live-cell imaging
15m
15m
Cells were cultured in 35 mm glass bottom dishes (MatTek).
HaloTag fluorescent ligands were added according to the manufacturer’s protocol (Promega).
After incubation for 00:15:00 in the incubator (37 °C and 5% CO2 ), the cells were quickly washed twice with PBS. The medium was replaced with Opti-MEM supplemented with 10% FBS.
15m
Imaging was performed using a Zeiss LSM900 confocal microscope in a temperature-controlled
(37 °C and 5% CO2) environment.