General protocol

Immunofluorescence

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

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

cytoskeletal buffer withouth Tritone-X for washes

compound	stock conc	final conc	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 μL	80 μL
MgCl2	1 M	3 mM	60 μL	120 μL
sucrose	2 M	300 mM	3	6
H2O			16.2	32.4
total			20	40

Pre-extraction buffer 2

0.2 % Triton X in PBS
note: more stringent; can be used for proteins bound to DNA

Fixing solution

4 % formaldehyde in PBS

Blocking solution

3 % BSA in PBS
Filter sterilize, before each usage inspect the solution for contamination. Filter again is necessary

Protocol

Cell seeding

1. insert sterile coverslips in dishes/well plates
2. seed the cells to have apporx. 70 % confluency (the next day or in the day of fixation)
3. optional: treatment, do not forget contols

Fixation without pre-extraction

1. optional: transfer coverslips with cells into multiwell plate
2. wash cells with r.t. 1x PBS
3. fix the cells with 500 uL fixing solution, leave for 15 min r.t. in the chemical hood
4. wash coverslips 3 times with 1x PBS
5. permeabilize coverslips with 0.2 % Triton-X in PBS for 10 min, r.t.
6. wash coverslips 2x wit 1x PBS

Fixation with CSK buffer pre-extraction

1. optional: transfer coverslips with cells into multiwell plate
2. wash cells with r.t. 1x PBS
3. add 500 uL of ice-cold pre-extraction buffer and leave it on ice for 5 - 10 minutes
4. wash the cells with ice-cold preextraction buffer without Triton-X
5. fix the cells with 500 μL fixing solution, leave for 15 min r.t. in the chemical hood
6. wash coverslips 3 times with 1x PBS
7. when staining PCNA: fix the cells addditionally with -20°C methanol (20 min at -20°C); wash 3x with 1x PBS

Staining

In humid (dark) chamber:

1. transfer coverslips into a humid chamber, cells facing up.
2. quickly add 100 uL 3% BSA in PBS on top of the coverslip, leave for 15 min, r.t. to block the cells. Avoid drying up of coverslips
3. wash coverslips 1x with 1xPBS, 1x with blocking solution
4. add primary antibodies, 50 uL on top of a coverslip, keep for 90 min in dark
5. wash coverslips 3 times with 1x PBS, roughly 200 uL
6. add secondary antibodies, 50 μL on top of a coverslip, keep for 30-60 min in dark
7. wash with 1x PBS
8. stain with DAPI, 1:2000 (1 μg/ml)