Núria Crua· Postdoc· University of Lausanne (UNIL)
Published Jul 27, 2026~5h 15m total· 3 contributions
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JNAR-ID: JNAR-89723045
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PublishedLast updated Jul 28, 2026
Objective
Immunofluorescent staining of HeLa cells for imaging, using a V5-tag primary antibody detected with an Alexa 488 anti-mouse secondary antibody, with DAPI and CellMask counterstaining.
Classification
Sample typeCells
TechniqueMicroscopy & ImagingCell Culture
Experimental contextIn vitro
Biological modelCell LinesHuman
Reproducibility status
Reproducible
No reproducibility blockers or risks reported. The protocol runs end-to-end.
21 healthy
⏱ Total 5 h 15 min1 section21 steps
Experimental Procedure
5 h 15 min
1
Harves the cells at the 70% of density on the glass-bottom culture support for the day of the experiment
2
Once the relevant treatments or incubations has finished, remove the medium.
3
Perform 3 gentle washes for 1 min with 1x PBS
4
Remove the medium and add 2 mL of freshly prepared 4% formaldehyde in 1x PBS to each sample.
TipIf needed, at this point you can store the samples in the fridge until the next day.
5
Incubate 15 minutes at room temperature (covered with aluminium foil).15 min
6
Remove the PFA and add 2 mL per sample of the permeabilization reagent (1% Triton X-100 in PBS).
7
Incubate 10 minutes at room temperature.10 min
8
Remove the permeabilization reagent and add 1 mL of the blocking solution (10% normal serum, 0.1% Triton X-100 in PBS).
9
Incubate 1 hour at room temperature in the dark.1 h
TipIf needed, samples can be left in the fridge until the next day in the same blocking solution.
10
Prepare the primary antibody at 1:50-200 (Depending of your primary antibody) in 1% BSA (or blocking reagent)
11
Add the primary antibody and incubate 2 hours at room temperature in the dark, with gentle shaking or 4º C overnight in a humidified chamber2 h
Critical stepThe sample must not dry out!
12
Wash 3 times for 10 minutes each with blocking solution without antibody.30 min
13
Prepare the secondary antibody diluted 1:500-1:1000 of blocking solution or 3% BSA
14
Add 500 µL of secondary antibody per sample (place it in the central part) and incubate 30 minutes at room temperature in the dark, with gentle shaking. 30 min
15
Wash 3 times for 10 minutes each with blocking solution without antibody.30 min
16
Prepare DAPI according to the manufacturer's instructions and add enough to cover the cells
17
Wash out the DAPI with 1x PBS for 5 minutes.5 min
18
Change medium and add CellMask add enough to cover the cells
19
Incubate 5-10 minutes in the dark.10 min
20
Wash out the CellMask with 1x PBS for 5 minutes.5 min
21
Add 15 µL of Mounting medium directly and place the coverslip.
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