Apr 03, 2023

Immunofluorescence staining

DOI
https://dx.doi.org/10.17504/protocols.io.36wgq7oq5vk5/v1
  • 1German Center for Neurodegenerative Diseases (DZNE), Tübingen, 72076 Germany
Hariam Raji
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DOI: https://dx.doi.org/10.17504/protocols.io.36wgq7oq5vk5/v1
Protocol Citation: michela.deleidi , Pascale Baden, Maria Jose Perez J., Hariam Raji, Federico Bertoli 2023. Immunofluorescence staining. protocols.io https://dx.doi.org/10.17504/protocols.io.36wgq7oq5vk5/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 25, 2022
Last Modified: May 31, 2024
Protocol Integer ID: 61418
Keywords: Fixation of the cells, HEK cells, neurons, Blocking and permeabilization, immunofluorescence Staining, ASAPCRN, immunofluorescence, cell
Abstract
This protocol describes the immunofluorescence staining of cells.
Attachments
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Materials
Recipes and products:
AB
8% Paraformaldehyde (PFA)
PFA20 g 
1M NaOH0.5 ml 
1x PBS100 ml
Note
  • Heat to ~60 °C to dilute. Then filter through folded filters into new cylinder.
  • Adjust 7.4 (normally its ~7.38 without adding something)
  • Fill up to 250 mL with 1x PBS.

Dako Mounting Medium Fluorescence Mounting MediumAgilent TechnologiesCatalog #S302380-2 :

NGS (normal-goat serum) Normal Goat Serum Blocking SolutionBIOZOLCatalog #VEC-S-1000
Note
  • Prepare aliquots out of stock and store in -20 °C .
  • Filter before use to avoid contamination.


DAPI (DAPI (4',6-Diamidino-2-Phenylindole, Dilactate) DAPI (46-Diamidino-2-Phenylindole Dilactate)BioLegendCatalog #422801 :
  • Dissolve the content in 2 mL deionized water (DAPI concentration 10.9 millimolar (mM) ).



Fixation of the cells: Strategy 1, i.e., HEK cells
25m
Remove medium.
Wash with 1× PBS.
Remove PBS and add 4% PFA to the cells.
Note
Note: For a coverslip in a 24 multi-well use at least 300 µL /well.


Incubate 00:10:00 at Room temperature .

10m
Collect PFA.
Wash 1× PBS.
Wash with 1× PBS for 00:05:00 at Room temperature . (1/3)

5m
Wash with 1× PBS for 00:05:00 at Room temperature . (2/3)
5m
Wash with 1× PBS for 00:05:00 at Room temperature . (3/3)
5m
Fixation of the cells: Strategy 2, i.e., neurons
25m
Add the same volume of 8% PFA as medium is in the well to the well.
Incubate 00:10:00 at Room temperature .
10m
Collect PFA in a falcon.
Wash in 1× PBS.
Wash with 1× PBS for 00:05:00 at Room temperature . (1/2)
5m
Wash with 1× PBS for 00:05:00 at Room temperature . (2/2)
Note
Storage until ICC: Keep coverslips in 1× PBS, seal the plate with parafilm and store at 4 °C .


5m
Blocking and permeabilization
25m
Remove 1X PBS.
Add 300 µL /well of blocking solution.
Note
Blocking solution: 10% NGS [normal goat serum] in PBS + Triton X-100 0,1%, filter the solution before using it.


Incubate at least 01:00:00 at Room temperature .

1h
Staining: Day 1
25m
Prepare antibody in blocking solution containing 5% NGS.
Put a drop (50 µL ) of Primary antibody solution on the parafilm surface.

Remove the coverslips from the plate and gently put it upside-down on the antibody drop.
Incubate Overnight at 4 °C .

1h
Staining: Day 2
25m
Wash:
Wash for 00:05:00 in PBS + Triton X-100 0.1%. (1/3)

5m
Wash for 00:05:00 in PBS + Triton X-100 0.1%. (2/3)
5m
Wash for 00:05:00 in PBS + Triton X-100 0.1%. (3/3)
5m
Prepare secondary antibody in blocking solution containing 5% NGS.
Note
Note: Keep in the dark.

Put a drop (50 µL ) of secondary antibody solution on the parafilm.

Take the coverslips and put it upside-down on the antibody drop.
Incubate 01:00:00 at Room temperature in the dark.

1h
Transfer the coverslip to a 24-well containing 500 µL 1X PBS + Triton X-100 0,1%.

Incubate for 00:05:00 at Room temperature (in the dark).

5m
Dilute DAPI 1:10000 in 1X PBS.
Remove PBS and incubate with DAPI for 00:05:00 at Room temperature in the dark.

5m
Wash.
Wash with 1X PBS. (1/2)
Wash with 1X PBS. (2/2)
Mount the slides:
Put a drop (10 µL ) of DAKO mounting reagent on the slide.

Take out the coverslip from the plate.
Dry it by gently tapping the coverslip’s edge on a lens-cleaner tissue.
Gently put the coverslips upside-down on the DAKO drop.
Leave it dry (24:00:00 , DARK, Room temperature ).

1d
Store in a slide-box at 4 °C .