Click reaction with EdU

Immunofluorescence

Material

Pre-extraction buffers

Fixing solution

4 % formaldehyde (PFA) in PBS

Blocking solution

3 % BSA in PBS

• filter sterilize, before each usage inspect the solution for contamination; filter again is necessary

Click reaction for EdU

for 500 μL
390 μL H2O
50 μL Tris.HCl pH 8
10 μL 100mM CuSO4
50 μL 1M Sodium Ascorbate
0.5 μL Alexa-azid

Protocol

Cell seeding

• insert sterile coverslips in dishes/well plates
• seed the cells to have approx. 70 % confluency day of experiment
• (add dox or other 24 h treatments)

Click reaction: + fixation without CSK buffer pre-extraction

• if the cells are seeded onto dish: transfer coverslips with cells into multiwell plate with pre-warm medium
• EdU staining: add EdU (final conc 20 μM; for 15 min)
• wash cells with PBS (r.t.)
• fix the cells with 500 μL of fixing solution (15 min r.t.; in the chemical hood)
• wash coverslips 3x with PBS
• permeabilize coverslips with 0.2 % Triton-X in PBS (10 min; r.t.)
• wash coverslips 2x with PBS

Click reaction: + fixation with CSK buffer pre-extraction

• optional: transfer coverslips with cells into multiwell plate
• treat the cells, for EdU staining add 20 μM of EdU for 15 min
• wash cells with r.t. 1x PBS
• add 500 uL of ice-cold pre-extraction buffer and leave it on ice for 5 or 10 minutes
• wash the cells with ice-cold preextraction buffer without Triton-X
• fix the cells with 500 μL fixing solution (15 min r.t. in the chemical hood)
• wash coverslips 3 times with 1x PBS
• 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)