Published Aug 14, 2026~1h 54m total· 0 contributions
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JNAR-ID: JNAR-17494362
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PublishedLast updated Aug 14, 2026
Objective
To perform immunofluorescence staining on cultured cells grown on coverslips, including fixation, permeabilization, antibody incubation, and mounting for microscopy.
No reproducibility blockers or risks reported. The protocol runs end-to-end.
13 healthy
⏱ Total 1 h 54 min2 sections13 steps
Preparation and Fixation
1
Remove the medium and wash with 1x PBS containing Ca and Mg.
2
Fix the cells as indicated in the cell fixation protocol.
3
Store at 4°C with PBS.
Critical step
TipFrom this point on, use PBS without Ca and Mg.
WarningNever use Falcon brand plates; the wells are too narrow for proper handling of coverslips.
Staining Procedure
1 h 54 min
4
Wash with PBS for 3 min (500 µl for 24-well plates and 300 µl for 48-well plates).3 min
5
Permeabilize with 0.1% Triton X-100 in PBS for 10 min at RT (10 µl in 10 ml PBS).10 min
6
Wash 2 times in PBS (3 min/wash).3 min
7
Block with Blocking Solution for 60 min at RT.
The blocking solution (10 ml) consisted of 10% (1 ml) goat serum, 0.1% (10 µl) Triton X-100, and 3% (3 ml) BSA diluted in PBS (5.99 ml).1 h
8
Remove the Blocking Solution and add the primary antibody dissolved in Antibody Solution. Use 50 µl of antibody for 12 mm coverslips and 30 µl for 8 mm coverslips. Incubate at 4°C o/n (overnight).
The antibody solution (10 ml) contained 1% (100 µl) goat serum, 0.1% (10 µl) Triton X-100, and 1% (1 ml) BSA diluted in PBS (8.89 ml).
Critical stepEnsure correct volume per coverslip size and proper sealing of the humidified chamber to prevent drying and uneven staining.
TipHow to set up the humidified chamber for the primary antibody: Use a plastic base and place filter paper on it. Moisten the paper without leaving excess water, ensuring you remove any bubbles. Place Parafilm on top, covering it entirely, and smooth out any bubbles between the paper and the Parafilm so the surface is completely level and the coverslip can adhere perfectly.
9
Carefully return the coverslips to the plate, making sure the cells are facing upwards, and wash 3 times with PBS (5 min/wash).5 min
Critical stepHandle coverslips gently to avoid dislodging cells or introducing air bubbles that could disrupt imaging.
10
Add the secondary antibody at 1:1000 (prepared in Antibody Solution) + DAPI at 1:10,000. Add 300 µl per well in a 24-well plate and 150 µl per well in a 48-well plate. Incubate for 30 min at RT with the plate covered in aluminum foil.
The antibody solution (10 ml) contained 1% (100 µl) goat serum, 0.1% (10 µl) Triton X-100, and 1% (1 ml) BSA diluted in PBS (8.89 ml).30 min
Critical step
WarningCover the plate with aluminum foil during incubation to protect fluorescent dyes from light exposure.
11
Wash 2 times with PBS (3 min/wash).3 min
12
Mount the coverslips on a microscope slide using MOWIOL. For 24-well plates (12 mm coverslips), use 5 µl of MOWIOL. (Briefly spin down the MOWIOL beforehand to remove any microbubbles).
Critical stepSpinning down MOWIOL prevents microbubbles that could obscure imaging. Ensure even application and proper orientation of the coverslip on the slide.
13
After mounting, leave them protected from light at RT to dry before storing them in their slide box at 4°C.
Critical step
WarningProtect mounted slides from light to preserve fluorescence signal.
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