Dec 03, 2024

Public workspaceImmunofluorescence - RAW 264.7

The Journal of Cell Biology
DOI
dx.doi.org/10.17504/protocols.io.36wgqd7wkvk5/v1
  • 1Department of Cell Biology, Yale University School of Medicine, New Haven, Connecticut 06510, USA;
  • 2Neuroscience, Yale University School of Medicine, New Haven, Connecticut 06510, USA;
  • 3Program in Cellular Neuroscience, Neurodegeneration and Repair;
  • 4Wu Tsai Institute Yale University School of Medicine, New Haven, Connecticut 06510, USA;
  • 5Aligning Science Across Parkinson’s (ASAP) Collaborative Research Network, Chevy Chase, MD, 20815, USA;
  • 6Kavli Institute for Neuroscience, Yale University School of Medicine, New Haven, Connecticut 06510, USA
Amanda Bentley-DeSousa
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DOI: dx.doi.org/10.17504/protocols.io.36wgqd7wkvk5/v1
Protocol Citation: Amanda Bentley-DeSousa, Shawn Ferguson 2024. Immunofluorescence - RAW 264.7. protocols.io https://dx.doi.org/10.17504/protocols.io.36wgqd7wkvk5/v1
Manuscript citation:
https://doi.org/10.1101/2023.10.31.564602
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: December 02, 2024
Last Modified: December 03, 2024
Protocol Integer ID: 113418
Keywords: RAW 264.7, immunofluorescence
Funders Acknowledgements:
Aligning Science Across Parkinson’s Disease
Grant ID: ASAP-000580
Abstract
This protocol details steps taken to perform immunofluorescence in RAW 264.7 cells.
Materials
DMEM (Thermo Fisher Scientific, 11965-092)
FBS (Thermo Fisher Scientific, 16140-071)
Penicillin/Streptomycin (Thermo Fisher Scientific, 15140122)
CellStripper (Corning, 356230)
PBS [1.1 mM KH2PO4 (J.T. Baker, 3246-01), 155.2 mM NaCl (Sigma-Aldrich, 3624-05), and 3 mM Na2HPO4 (J.T. Baker, 3828-05)]
Paraformaldehyde (Electron Microscopy Sciences, 19202)
Sodium phosphate buffer (pH 7.3 / Buffer: 153.56 mM Sodium phosphate, dibasic, anhydrous, J.T. Baker 3828, 53.63 mM Sodium dihydrogen phosphate monohydrate, J.T. Baker 3818)
Sucrose (Sigma-Aldrich, S0389)
BSA (Sigma-Aldrich, A9647)
Saponin (Sigma-Aldrich, S4521)
Triton (Sigma-Aldrich, X100)
Coverslips (12 mm, Carolina Biological Supplies, 633029)
Microscope Slides (Thermo Fisher Scientific, 12-550-143)
Prolong Gold Mounting Media (Thermo Fisher Scientific, P36935)
Day 0
Day 0
Plate 2 x 10^5 RAW 264.7 cells on poly-D-lysine coated coverslips.
Day 1
Day 1
35m
35m
Add the indicated compound at the provided concentration and time point in fresh DMEM media containing FBS and P/S.
Proceed to next step if not treating cells.
Fix cells in 4% paraformaldehyde supplemented with 4% sucrose for Duration00:30:00 atTemperatureRoom temperature .

30m
Wash coverslips 3X for Duration00:05:00 with PBS at TemperatureRoom temperature .

5m
Add primary antibodies DurationOvernight at Temperature4 °C in 3% BSA in PBS.

Day 2
Day 2
1h 10m
1h 10m
Wash coverslips 3X for Duration00:05:00 with PBS at room temperature.

5m
Add secondary antibody for Duration01:00:00 at TemperatureRoom temperature in PBS in the dark.

1h
Wash coverslips 3X for Duration00:05:00 with PBS at TemperatureRoom temperature .

5m
Mount coverslips onto microscope slides with Prolong Gold mounting media and store at Temperature4 °C .

Day 3+
Day 3+
Image coverslips.