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Materials

  • EdU kit baseclick: -20◦C; 2.14; ;chemicals box
  • EdU kit baseclick: 4◦C; 2.14; 4F44; kitsBoxes
  • PBS
  • 4% PFA

Day1

EdU preparation

  • thaw DMSO diluted stock (10mM)
  • dilute in PBS to 1mM -> working solution

Cells labelling

  • add working solution in ratio 1:100 in the wells with media
  • incubate 30 min. at 37◦C

Cells fixation

  • move to chemical cabinet in genomic room
  • aspirate media
  • wash with PBS
  • fix with 4% PFA for 10 min. at RT
  • aspirate
  • wash with PBS
  • aspirate
  • add PBS for storage
  • store at 4◦C

Cell labelling EdU kit

  • Aspirate PBS
  • Permeabilze with 0.1% Triton X-100 in PBS for 20 minutes at RT
  • Prepare the EdU staining solution (should be used within 15 min)
  • Remove permeabilization solution
  • Wash 2 x with 3% BSA in PBS
  • Aspirate
  • Add EdU staining solution incubate for 30 minutes in dark
  • From now on avoid exposure to light
  • Remove EdU staining solution
  • Wash 3 x with 3% BSA in PBS
  • Aspirate
  • Add PBS
  • Note: You can stop here and store the cells at 4◦C

Staining with primary antibodies

  • Aspirate PBS
  • Add blocking solution (1% BSA in 0.05% Tween-20 PBS) and incubate for 30 minutes
  • Aspirate
  • Add antibodies diluted in 1% BSA in 0.05% Tween-20 PBS
  • Keep in dark at 4◦C overnight

Day 2

Note: Limit exposure to direct light

Solutions to prepare

  • 0.05% Tween 20 in PBS (for 3 washes and antibody incubation)
  • Solution of secondary antibody and phalloidine in 0.05% Tween 20 in PBS (together)
  • DAPI in PBS (1:5000) (make 10mL stock)
  • PBS
  • Mowiol

Secondary antibodies incubation

  • Aspirate primary anitbody.
  • Wash with 0.05% Tween 20 in PBS (3x)
  • Prepare secondary antibody and phalloidin
  • Add secondary antibody to the wells
  • Incubate for 1h with secondary antibody and phalloidin
  • Aspirate
  • Wash 1x with PBS

Dapi counterstaining

  • Add DAPI working solution to stain nuclei
  • Incubate 5min
  • Aspirate
  • Wash 3x 5 min with PBS
  • Check under microscope
  • Mount in Mowiol
  • Store at 4◦C in dark

Notes

  • Phalloidine staining does not work with EdU staining
  • Primary antibodies:
  • Secondary antibodies (1:400):
  • Phalloidine(1:100):