May 24, 2023
Immunofluorescence assay (IFA)
- Thanh Ngoc Nguyen1
- 1Laboratory of Michael Lazarou, Walter and Eliza Hall Institute of Medical Research, Parkville, Victoria, Australia
- Thanh Ngoc Nguyen: nguyen.tha@wehi.edu.au
Protocol Citation: Thanh Ngoc Nguyen 2023. Immunofluorescence assay (IFA). protocols.io https://dx.doi.org/10.17504/protocols.io.5qpvobz99l4o/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: July 19, 2022
Last Modified: May 31, 2024
Protocol Integer ID: 67016
Keywords: Immunofluorescence, HistoGrip, Permeabilization, ASAPCRN
Funders Acknowledgement:
Aligning Science Across Parkinson’s
Grant ID: ASAP-000350
Abstract
This protocol details the procedure of immunofluorescence assay (IFA).
Attachments
ihdnbr7hf.docx
16KB
Materials
Buffers:
- 1x PBS
- Fixation buffer: 4% (w/v) paraformaldehyde (PFA) in 0.1 Molarity (M) phosphate buffer.
- Permeabilization buffer: 0.1 TritonX-100/1xPBS
- Blocking buffer: 3% Goat serum in 0.1 Tween20/1xPBS
Mounting medium:
| A | B | |
| DABCO | 0.2M | |
| Tris-Cl pH 8.0 | 80 mM | |
| Glycerol | 90% |
14-Diazabicyclo[2.2.2]octaneSigma-aldrichCatalog #D27802
- Dissolve 0.23 g DABCO in 200 µL H2O.
- Add 0.8 mL 1 Molarity (M) Tris-Cl pH 8.0.
- 9.0 mL glycerol and mix on rotary shaker.
- Store at -20 °C wrapped in foil.
Procedures
Procedures
2d 6h 35m
2d 6h 35m
Seed the HeLa cells onto HistoGrip (ThermoFisher) coated coverslips in growth media in 6 well plates (DMEM supplemented with 10 % (v/v) FBS, 1 % Penicillin-Streptomycin, 25 millimolar (mM) HEPES, GlutaMAX (Life Technologies) and non-essential amino acids (Life Technologies)).
Note
After 48:00:00 , cells can be subjected to any required treatment prior to fixation.
Aspirate off the culture media and add 700 µL of fixation buffer to each well and incubate on a side-to-side shaker for 00:10:00 .
10m
Aspirate off the fixation solution and wash three times with 1x PBS.
Permeabilise the cells with Permeabilization buffer (2 mL for each well) for 00:10:00 on a side-to-side shaker.
10m
Aspirate off the Permeabilization buffer and wash once with 1x PBS.
Aspirate off PBS and incubate the cells with Blocking buffer (700 µL for each well) for 00:15:00 on a side-to-side shaker.
15m
Aspirate off the Blocking buffer, wash once with 1x PBS and once with permeabilization buffer.
Aspirate off the permeabilization completely and put the 6 well plates on a flat bench.
Add the primary antibodies (diluted to the right concentrations in Blocking buffer; 15 µL /well) onto the glass coverslips and incubate for 01:00:00 - 02:00:00 depending on the strength of the antibodies.
3h
Wash the coverslips twice with 1x PBS and once with permeabilization buffer.
Aspirate off the permeabilization completely and put the 6 well plates on a flat bench.
Add the relevant secondary antibodies (diluted in Blocking buffer to 1/200-1/500; 15 µL /well) onto the glass coverslips and incubate in the dark for 01:00:00 – 02:00:00 .
3h
Wash three times with 1x PBS and mount the coverslips on microscope slides.
Use a yellow tip, drop one small drop off mounting medium onto a microscope slide.
Pick out one coverslip from a well (still in 1x PBS) and place it onto a piece of whatman paper so that the side that the cells grew on is facing up.
Take the microscope slide with a drop of mounting medium and put it on the coverslip and gently push it down
to remove excessive mounting medium.
Seal the edges with nail polish, let it dry and store in a microscope box at 4 °C prior to imaging.