Jul 03, 2024
Immunofluorescence for adherent cells
- Alexandros C Kokotos1,2,
- Tim Ryan1,2
- 1Department of Biochemistry, Weill Cornell Medicine, New York, NY 10065, USA;
- 2Aligning Science Across Parkinson's (ASAP) Collaborative Research Network, Chevy Chase, Maryland 20815, USA
Protocol Citation: Alexandros C Kokotos, Tim Ryan 2024. Immunofluorescence for adherent cells. protocols.io https://dx.doi.org/10.17504/protocols.io.ewov19z5olr2/v1
Manuscript citation:
Phosphoglycerate kinase is a central leverage point in Parkinson’s Disease driven neuronal metabolic deficits
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 03, 2024
Last Modified: July 03, 2024
Protocol Integer ID: 102821
Keywords: ASAPCRN, immunofluorescence, immunostaining, immunocytochemistry, antibodies
Funders Acknowledgement:
ASAP
Grant ID: 000580
Abstract
This protocol describes a method to perform immunostaining for adherent cells.
Guidelines
Cells should be cultured and handled following all local biosafety regulations.
Materials
Equipment
Inverted tweezers (like Roboz RS-5020)
Consumables
PBS (Sigma Aldrich P3813) supplemented with 0.01 % sodium azide (Sigma Aldrich S-8032)
4 % PFA in PBS solution (Thermo Scientific J19943-K2)
NH4Cl (Sigma Aldrich A4514)
Triton X-100 (Sigma Aldrich T8787)
BSA (Sigma Aldrich A4503)
Parafilm (Electron Microscopy Sciences 70990)
Glass slides (VWR 16004-368)
Prolong Diamond mountant (Thermo Scientific P36970)
Clear nail polish
Before start
This protocol describes a method to perform immunofluorescence on adherent cells on coverslips. It can also be used for immunocytochemistry by changing the secondary antibodies and also adjusted for cells growing on plastic. This protocol has been verified for use with both cell line cells and primary cells, like neurons.
Immunofluorescence
Immunofluorescence
4h
Fix cells with 4 % (v/v) PFA in PBS for 00:10:00 .
Note: Discard PFA in a special container and not in the drain.
10m
Quench PFA by using a 50 millimolar (mM) NH4Cl solution in PBS for 00:10:00 .
10m
Permeabilize cells with 0.25 % (v/v) Triton X-100 in PBS for 00:10:00 .
10m
Block with 4 % (v/v) BSA in PBS for 01:00:00 .
Note: At this step the protocol can be paused. Blocking can also be done overnight.
Other blocking solutions can be used, such as goat or donkey serum matching with the species of the secondary antibodies used.
1h
Incubate primary antibodies in 4 % (v/v) BSA in PBS for 01:00:00 .
For adherent cells on glass coverslips, the antibody solution is drop seeded on parafilm and the coverslip is placed upside down on the antibody solution using the inverted tweezers. For the 22x22 mm coverslips, 100 ul of antibody solution is sufficient for the whole coverslip to be incubated with antibody. For cells growing on plastic the antibody solution can be added in the well/dish.
1h
Wash cells with PBS for 00:05:00 three times.
15m
Incubate secondary antibodies in 4 % (v/v) BSA in PBS for 01:00:00 .
For immunofluorescence, we usually use Alexa dye secondary antibodies (Thermo Scientific) at a 1:500 dilution.
1h
Wash cells with PBS for 00:05:00 three times.
15m
Wash cells with ddH2O to remove excess salt and protein crystals.
For cells on coverslips, the coverslips are dipped into a beaker with ddH2O four times and excess ddH2O is removed by touching the edge of the coverslips on absorbent paper.
Add the coverslip on a glass slide that has been previously drop seeded with Prolong Diamond mountant. Place coverslips inverted and avoid trapping any air in the process. Allow to air dry overnight in a dark environment.
For the 22x22 mm coverslips, 50 ul of mountant is sufficient. Pipette mountant with a cut tip to avoid bubbles.
Seal the coverslips the next day using clear nail polish.