General protocol: Immunofluorescence

General protocol and pre-extraction.

Posted Jan 31, 2025 Updated Apr 12, 2026

By Martin , Šárka

6 min read

Size of well
bigger wells: better for handling with CVs, more reagents and solutions (more user friendly)
6-well plate: 1 mL of solutions, 2 mL of PBS for washes
12-well plate: 0.5 - 1 mL of solutions, 1 - 2 mL of PBS for washes
24-well plate: 0.5 mL of solutions, 1 mL of 1xPBS for washes (the most difficult for handling with CVs)

The protocol might be modify for specific cell lines or proteins;
works well for U2OS, Hela, RPE-1 or MDF (mouse dermal fibroblast).

Wash general
aspirate liquid
add washing solution
aspirate liquid
…… repeat ……
add desire liquid

Wash during IF
in wells: in each step put CVs back for wash, more solvent help to reduce contain of previous reagents
on parafilm: once you place CVs on parafilm just fill CVs with washing solution
in boht cases: can be done on shaking platform to improve wash

Size of well
bigger wells: better for handling with CVs, more reagents and solutions (more user friendly)
6-well plate: 1 mL of solutions, 2 mL of PBS for washes
12-well plate: 0.5 - 1 mL of solutions, 1 - 2 mL of PBS for washes
24-well plate: 0.5 mL of solutions, 1 mL of 1xPBS for washes (the most difficult for handling with CVs)

Protocol

Cell seeding

insert sterile coverslips in dishes/well plates
seed the cells to have approx. 70 % confluency (the next day or in the day of fixation);
seeding HeLa, U2OS, DMF etc. 20 000 - 30 000 cells/cm²

Note: treatment, do not forget contols

Seeding to have homogenous distrubution of cells:
prepare stock of desire cells concentration
place all coverslips into sufficient plate (often 6-cm plate)
add add appropriate volume of cells’ stock
when cells are attached or day of treatment: tranfer coverslips in individual wells

❀ Prepare ❀

❀ pre-warm: DMEM, PBS for treatment and CVs transfer to wells
❀ prepared all materials, reagents, solutions and treatment

Before IF or treatment
Transfer coverslips into well plate

add pre-warm medium or PBS to each well
transfer CVs with tweezer (if you can not lift it: try to lift if with P200 or P2 tip agains plate wall with one hand and grab it with tweezer in other hand)
… continue with treatment/IF …

Pre-extraction

Removing soluble, non-chromatin-associated proteins before fixing the cell sample; reducing background noise and improving the signal-to-noise ratio; particularly for nuclear proteins.

wash cells with PBS (1-2x)
ice-cold pre-extraction buffer (5-10 min; on ice)
wash the cells with preextraction buffer without Triton-X (2x)
Example:
- 0.2 % Triton X in PBS
- Note*: more stringent; can be used for proteins bound to DNA
- cytoskeletal (CSK) buffer
- methanol (-20°C) pre-extraction & fixation; wash only PBS

Fixation

The preservation of biological tissues from decay. It terminates any ongoing biochemical reactions; may also increase mechanical strength or stability.

w/o pre-extraction: wash cells with PBS (2x)
fix the cells with fixing solution
wash with 1x PBS (3 x 5 min)
Example:
- 4% Formaldehyde (15 min; r.t.; work in the chemical hood)
- methanol: (20 min; -20°C)

Blocking solution

Blocking: Reduce non-specific background noise by covering exposed binding sites.

option 1: in wells
option 2: place CVs on parafilm into wet chamber; add antibody solution; cells facing up; approx. 200 μL/CV
incubation (30 min; r.t.)

Example:
3 % BSA in PBS
Note: filter sterilize, before each usage inspect the solution for contamination; filter again is necessary
DMEM with 10% FBS
10% FBS-PBS

❀ In mean time ❀

❀ prepare wet chamber
❀ dilute primary antibody

Staining: primary antibodies

optional: drain slides briefly dipping on a tissue, but avoid drying up of CVs
dilute the antibodies in blocking solution (mix all in the one ependorph)
dilution according to specific protocol
incubation (1 - 2 h; r.t.; wet chamber)
option 1 - easier:
50 μL/CV: place CVs on parafilm into wet chamber; add antibody solution; cells facing up
option 2:
20 - 30 μL/CV: add antibody solution; place CVs on parafilm into wet chamber - flip CVs; cells facing down
after incubation for washes, place CVs on parafilm into wet chamber or wells - flip CVs again; cells facing up
option 3 - incubation over night:
20 - 30 μL/CV: add antibody solution; place CVs on parafilm into wet chamber - flip CVs; cells facing down
incubation (O/N; 4°C; wet chamber)
place CVs on parafilm into wet chamber or wells - flip CVs again; cells facing up

wash CVs with PBS (3 x 5 min; approx. 200 μL/CV)

Staining: secondary antibodies

Choosing of secondary antibodies
Based on their excitation and emission spectrum Thermofisher: Fluorescence SpectraViewer

Example:
DAPI: emission 461 nm
Green Fluorescent Protein (GFP): emission 488 nm
PCNA: has to have different emission (wavelengths), e.g. 555 nm, 647 nm

Secondary antibody & samples have to be kept in dark from now on. Use dark wet chamber for further staining and work.

optional: drain slides briefly dipping on a tissue, but avoid drying up of CVs
dilute the antibodies in blocking solution (mix all in the one ependorph)
dilution according to specific protocol
option 1 - easier: 50 μL/CV: place CVs on parafilm into dark wet chamber; add antibody solution; cells facing up
option 2: 20 - 30 μL/CV: add antibody solution; place CVs on parafilm into dark wet chamber - flip CVs; cells facing down
incubation (30 - 60 min; r.t.; wet chamber)
option 2: for washes after incubation, place CVs on parafilm into wet chamber or wells - flip CVs again; cells facing up

wash CVs with PBS (3 x 5 min; approx. 200 μL/CV)

Staining: DNA content

Example:
Hoechst: before IF stainig; add directly to DMEM; (1μg/mL; 30 min; 37°C; dark; e.g. incubator in cell culture room)
Hoechst: as DAPI at the end of IF (1μg/mL; 30 min; dark)
DAPI, 1:2000 (1 μg/ml)
DAPI in mount media

wash CVs with PBS (2 x 5 min; approx. 200 μL/CV)
wash CVs with H₂O (dipinto H₂O in beaker or dish/well)
dip to tissue to remove excess of liquid
air dry (approx. 30 min; r.t.; in dark; cells facing down)

Mounting of CVs

Mounting Guide Thermofisher: Overview of antifade mounting medium
Vectorlabs: Immunofluorescence Mounting Guide

3 – 4.5 μl/CV (cells facing down)
non-hardering: sealing CVs with nailpolich - apply around each CVs
hardering: sealing CVs with nailpolich - not necessary, but recomended especially for hight throuput microscopy
air dry (r.t.; in dark)

Material

Pre-extraction buffer 1

CSK (cytoskeletal) buffer

compound	stock concentration	final concentration	volume [mL]	volume [mL]
HEPES	1 M; pH 7.7	25 mM	0.5	1
NaCl	5 M	50 mM	0.2	0.4
EDTA	0.5 M	1 mM	40 uL	80 uL
MgCl₂	1 M	3 mM	60 μL	120 μL
sucrose	2 M	300 mM	3	6
TritonX	10 %	0.5 %	1	2
H₂O			15.2	30.4
total			20	40

Protocols, Immunofluorescence

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