Oct 13, 2025
Immunofluorescence staining
Forked from Immunofluorescence staining
- Devin Fuller1,
- Yumei Wu1,
- Florian Schueder1,
- Burha Rasool1,
- Shanta Nag1,
- Justin L. Korfhage1,
- Rolando Garcia-Milian1,
- Katerina D. Melnyk1,
- Joerg Bewersdorf1,
- Pietro De Camilli1,
- Thomas Melia1
- 1Yale University
Protocol Citation: Devin Fuller, Yumei Wu, Florian Schueder, Burha Rasool, Shanta Nag, Justin L. Korfhage, Rolando Garcia-Milian, Katerina D. Melnyk, Joerg Bewersdorf, Pietro De Camilli, Thomas Melia 2025. Immunofluorescence staining. protocols.io https://dx.doi.org/10.17504/protocols.io.5jyl8e5p8l2w/v1
Manuscript citation:
Fuller Devin M, Wu Yumei, Schueder Florian, Rasool Burha, Nag Shanta, Korfhage Justin L, Garcia-Milian Rolando, Melnyk Katerina D, Bewersdorf Joerg, De Camilli Pietro, Melia Thomas J (2025) ATG2A engages RAB1A and ARFGAP1 positive membranes during autophagosome biogenesis eLife 14:RP107316
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: April 07, 2025
Last Modified: October 13, 2025
Protocol Integer ID: 126304
Keywords: Fixation of the cells, HEK cells, neurons, Blocking and permeabilization, immunofluorescence Staining, ASAPCRN, immunofluorescence, cell
Funders Acknowledgements:
National Institutes of Health
Grant ID: R01 GM100930
National Institutes of Health
Grant ID: R35 GM153482
National Institutes of Health
Grant ID: DA018343
National Institutes of Health
Grant ID: F31 AG079606
National Institutes of Health
Grant ID: F31 DK136246
Aligning Science Across Parkinson’s
Grant ID: ASAP-025173
Human Frontier Science Program
Grant ID: LT000056/2020-C
Abstract
This protocol describes the immunofluorescence staining of cells.
Materials
Cell culture materials:
DMEM (Thermo Fisher Scientific, 11965-092)
FBS (Thermo Fisher Scientific, 16140-071)
Penicillin/Streptomicin (10,000 U/mL; Thermo Fisher Scientific, 15140122)
PBS (Thermo Fisher Scientific, 10010023)
Earle’s Balanced Salt Solution (EBSS; Thermo Fisher Scientific, 24010043)
16% Paraformaldehyde (Electron Microscopy Sciences, 15710)
Bovine serum albumin (BSA; Sigma-Aldrich, A9647)
Saponin (Sigma-Aldrich, 47036-50G)
Fluoromount-G (SouthernBiotech, 0100-01)
Troubleshooting
Fixation of HEK293 cells in 24 well plate
35m
Remove medium and add 4% PFA to the cells.
Note
Note: For a coverslip in a 24 well plate use at least 300 µL /well.
Incubate 00:20:00 at Room temperature . For cells expressing light sensitive fluorescent proteins, cover the 24 well plate with aluminum foil to block out ambient light.
20m
Collect PFA.
Perform the following washes with 1× PBS:
Wash with 1× PBS for 00:05:00 at Room temperature . (1/3)
5m
Wash with 1× PBS for 00:05:00 at Room temperature . (2/3)
5m
Wash with 1× PBS for 00:05:00 at Room temperature . (3/3)
5m
Blocking and permeabilization
15m
Remove 1X PBS.
Add 300 µL /well of blocking solution.
Note
Blocking solution: 5% BSA [Bovine Serum Albumin] in 1× PBS + 0.1% Saponin
Incubate at least 00:15:00 at Room temperature .
15m
Staining: Day 1
25m
Prepare primary antibody in blocking solution (typically 1:500, but can vary based on antibody staining).
Put a drop (50 µL ) of Primary antibody solution on the parafilm surface.
Remove the coverslips from the plate and gently put it upside-down on the antibody drop.
Alternatively, leave the coverslips in the well and add (250 µL ) of the primary antibody solution to each well.
Incubate Overnight at 4 °C .
1h
Staining: Day 2
25m
Perform the following washes:
Wash for 00:05:00 in blocking solution (1/3)
5m
Wash for 00:05:00 in blocking solution. (2/3)
5m
Wash for 00:05:00 in blocking solution. (3/3)
5m
Prepare secondary antibody in blocking solution (typically 1:500, but can vary based on antibody staining).
Note
Note: Keep in the dark. Keep the plate covered in aluminum foil if not doing so previously.
Put a drop (50 µL ) of secondary antibody solution on the parafilm.
Take the coverslips and put it upside-down on the antibody drop.
Alternatively, leave the coverslips in the well and add (250 µL ) of the secondary antibody solution to each well.
Incubate 01:00:00 at Room temperature in the dark (covered in foil).
1h
Perform the following washes:
Wash for 00:05:00 in blocking solution (1/3)
Wash for 00:05:00 in blocking solution. (2/3)
Wash for 00:05:00 in blocking solution. (3/3)
Mount the slides:
Put a drop (10 µL ) of Fluoromount-G on the slide.
Take out the coverslip from the plate.
Dry it by gently tapping the coverslip’s edge on a kimwipe.
Gently put the coverslips upside-down on the Fluoromount-G drop.
Leave it dry (24:00:00 , DARK, Room temperature ).
1d
Apply clear nail polish around the coverslips to ensure rigidity of the mount.
Store in a slide-box at 4 °C .
Acknowledgements
This work was supported by grants from the National Institutes of Health (R01 GM100930 and R35 GM153482 to TJM; R01 GM151829 to JB; DA018343 to PDC), F31 AG079606 to DMF and F31 DK136246 to JLK. This research was also funded in part through Aligning Science Across Parkinson’s (ASAP-025173 to TJM and PDC) through the Michael J. Fox Foundation for Parkinson’s Research (MJFF) and the Howard Hughes Medical Institute (HHMI; PDC). FS acknowledges support from the Human Frontier Science Program (LT000056/2020-C). JB acknowledges support by the Wellcome Leap Foundation. Imaging was supported by the Yale Center for Cellular and Molecular Imaging (both the fluorescence and electron microscopy facilities). We also thank the MS & Proteomics Resource at Yale University for providing the necessary mass spectrometers and the accompany biotechnology tools funded in part by the Yale School of Medicine and by the Office of The Director, National Institutes of Health (S10OD02365101A1, S10OD019967, and S10OD018034). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.