Apr 09, 2025
Immunofluorescence HeLa cells
- Francesca Filippini1
- 1Yale University
Protocol Citation: Francesca Filippini 2025. Immunofluorescence HeLa cells. protocols.io https://dx.doi.org/10.17504/protocols.io.n2bvjdxrwvk5/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: April 04, 2025
Last Modified: April 09, 2025
Protocol Integer ID: 126187
Abstract
Immunofluorescence HeLa cells with BSA/saponin
Day 0
Day 0
Plate 1,8 x 10^5 HeLa cells on 12 mm coverslips.
Day 1
Day 1
3h
3h
Transfect with indicated transfection reagent in DMEM media containing FBS and P/S.
Proceed to next step if not transfecting cells.
Fix cells adding 8% paraformaldehyde directly into culture media in 1:1 ratio to obtain 4% final concentration of paraformaldehyde.
30m
Block and permeabilize cells in PBS/0.1% Saponin/3% BSA for at least 00:15:00 atRoom temperature
15m
Wash coverslips 3X for 00:05:00 with PBS at Room temperature
5m
Apply primary antibody diluted in PBS/0.1% Saponin/3%BSA and incubate 01:00:00 at Room temperature or Overnight at 4 °C .
1h
Wash 3X for 00:05:00 with PBS/01% Saponin/3% BSA.
5m
Apply Alexa Fluor secondary antibodies (1:600 from 1 mg/ml stock) diluted in PBS/0.1% Saponin/3% BSA for 01:00:00 at Room temperature in the dark.
1h
Wash coverslips 3X for 00:05:00 at with PBS/01% Saponin/3% BSA.
5m
Mount coverslips on microscope slides with Prolong Gold mounting medium.
Allow to dry o/n at Room temperature in the dark. Store at 4 °C
Day 3
Day 3
Image coverslips at microscope.