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Preprint

Immunofluorescence

KS Kristine Schauer
MC Mathieu Coppey
KV Kotryna Vaidziulyte

Last updated date: Sep 20, 2022 Views: 528 Forks: 0

original An abbreviated version of this protocol was published in eLife in Mar, 2022
Persistent cell migration emerges from a coupling between protrusion dynamics and polarized trafficking
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  1. Coverslip preparation
    1. round glass coverslips (d=15 mm, #1) were plasma cleaned (2 min vacuum, 1 min plasma)
    2. cleaned coverslips were coated with fibronectin (2 ug/mL) in 100 mM NaHCO3 (pH 8.5) for 1h in room temperature
    3. coated coverslips were washed with PBS (1x) three times. (Prepared coverslips can be stored in +4C in PBS (1x) for at least a week.)
  2. Cell seeding
    1. cells were seeded on coverslips (placed in a 12-well plate) 48h before the start of the experiment and left to attach and spread.
  3. Fixation
    1. aspiration of growth medium
    2. fixation with cold (-20C) methanol
    3. rehydration with PBST (PBS-Tween) buffer (x3 for 5min)
    4. blocking with 3% BSA in PBST buffer for 1h at room temperature (RT)
    5. incubation with primary antibodies against a-Tubulin (mouse, T5168 Clone B-5-1-2, dilution 1:1000) for 40 min at RT
    6. washing with with PBST buffer (x3 for 5min)
    7. incubation with secondary antibodies against mouse (AlexaFluor 546 F(ab’)2 fragment of goat anti mouse IgG (H + L), dilution 1:1000) for 30 min at RT
    8. washing with with PBST buffer (x3 for 5min)
    9. nuclei staining with DAPI (1 ug/mL) for 15 min at RT
    10. washing with with PBST buffer (x3 for 5min)
    11. coverslips were drained on a Kimwipe and mounted on a drop of Fluoromount placed on a rectangular glass slide
    12. after overnight incubation in +4C, coverslips were sealed with nail polish, cleaned and imaged
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How to cite：
Readers should cite both the Bio-protocol preprint and the original research article where this protocol was used:
  1. Schauer, K, Coppey, M and Vaidziulyte, K(2022). Immunofluorescence. Bio-protocol Preprint. bio-protocol.org/prep1955.
  2. Vaidžiulytė, K., Macé, A., Battistella, A., Beng, W., Schauer, K. and Coppey, M.(2022). Persistent cell migration emerges from a coupling between protrusion dynamics and polarized trafficking. eLife. DOI: 10.7554/eLife.69229

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