Jul 07, 2023
Immunofluorescence of macrophages and microglia
- Narayana Yadavalli1,2,3,4,5,
- Shawn M. Ferguson1,2,3,4,6,5
- 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
Protocol Citation: Narayana Yadavalli, Shawn M. Ferguson 2023. Immunofluorescence of macrophages and microglia. protocols.io https://dx.doi.org/10.17504/protocols.io.81wgbym7ovpk/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: May 25, 2023
Last Modified: May 31, 2024
Protocol Integer ID: 82457
Keywords: Immunofluorescence, antibody, ASAPCRN
Funders Acknowledgement:
ASAP
Grant ID: 000580
Abstract
This protocol describes the fixation and staining of cultured cells in paraformaldehyde solution.
Attachments
mqjvbj3up.docx
16KB
Materials
Solutions to prepare
Fixative solution: 4% paraformaldehyde in 0.2 M phosphate buffer pH 7.4.
Permeabilization and locking solution: 0.1% (v/v) saponin + 5% (w/v) BSA in PBS
Primary antibody solution: 0.1% (v/v) saponin + 5% (w/v) BSA in PBS solution
Secondary antibody solution: 0.1% (v/v) saponin + 5% (w/v) BSA in PBS solution
LAMP-1 (human)Developmental Studies Hybridoma BankCatalog #ID4B
Anti-TFE3 antibody produced in rabbitMerck MilliporeSigma (Sigma-Aldrich)Catalog #HPA023881
ProLong™ Gold Antifade Mountant with DAPIThermo FisherCatalog #P36935
Immunofluorescence of macrophages and microglia
Immunofluorescence of macrophages and microglia
3h 55m
3h 55m
Plate 50,000 iPSC derived macrophages or microglia or Neurons or BMDM’s cells on 22 x 22 mm glass coverslips in 24-well dishes in respective culture media.
Incubate Overnight at 37 °C in 5% CO2.
Pre-warm a solution of 4% paraformaldehyde to Room temperature .
Aspirate media from cells and immediately proceed to step 5.
Add pre-warmed 4% paraformaldehyde to cells.
Incubate for 00:15:00 at Room temperature .
15m
Remove 4% paraformaldehyde solution and dispose of it in an appropriate chemical waste container.
Rinse each well with 3X 1 mL PBS. Remove rinse and dispose of it in an appropriate chemical waste container.
Add 0.1% (v/v) saponin + 5% (w/v) BSA in PBS and incubate for 01:00:00 at Room temperature to permeabilize cells.
1h
Aspirate 0.1% (v/v) saponin + 5% (w/v) BSA in PBS solution.
Prepare respective antibody dilutions in 0.1% (v/v) saponin + 5% (w/v) BSA in PBS solution. mouse LAMP1 (DSHB #ID4B) 1:200 dilution, TFE3 (Sigma #HPA023881), 1:200 dilution.
Incubate at 4 °C Overnight .
1h
Aspirate antibody solution and wash cells with PBS for 00:05:00 .
5m
Repeat wash.
Wash with PBS for 00:05:00 . (1/3)
5m
Wash with PBS for 00:05:00 .(2/3)
5m
Wash with PBS for 00:05:00 .(3/3)
5m
Add filtered PBS containing 5% BSA and secondary antibodies (1:600, Alexa fluorophores 488 and 555, Invitrogen).
Incubate at Room temperature for 01:00:00 .
1h
Aspirate antibody solution and wash cells.
Wash cells with PBS for 00:05:00 . (1/3)
5m
Wash cells with PBS for 00:05:00 . (2/3)
5m
Wash cells with PBS for 00:05:00 . (3/3)
5m
Rinse coverslips in milliQ water.
Mount coverslips onto slides using ProLong Gold Antifade Mountant with DAPI (ThermoFisher P36935).
Allow slides to cure Overnight in darkness.
5m
Image.