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Immunofluorescence Detection and F-actin Staining of MTLn3 Cells

HP Huan Pang

Published: Apr 20, 2012 DOI: 10.21769/BioProtoc.206 Views: 15574

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Abstract

Epidermal growth factor (EGF)-stimulated MTLn3 cells protrusion play an important role in cell migration. Phalloidin which binds F-actin in cells is an imaging tool used with light microscopy to investigate the distribution of actin. The protocol described here can be useful for observing signaling in the EGF pathway.

Background

Materials and Reagents

  1. MTLn3 cells
  2. Type I rat tail collagen (BD Biosciences, catalog number: 354236 )
  3. Leibovitz's L15 media w/o phenol red (Life Technologies, InvitrogenTM, catalog number: 21083-027 )
  4. Leibovitz's L15 media w/ phenol red (Life Technologies, InvitrogenTM, catalog number: 11415-064 )
  5. Rhodamin phalloidin red Cy3 (Life Technologies, InvitrogenTM, catalog number: R415 )
  6. Phosphate buffered saline (PBS)
  7. Trypsin-EDTA
  8. HCI
  9. 95% ethanol
  10. Acetic acid
  11. FBS-alpha-MEM
  12. Paraformaldehyde
  13. Triton X-100
  14. Glycine
  15. BSA
  16. N-propyl gallate
  17. Glycerol
  18. Methanol
  19. Nail polish
  20. Collagen solution
  21. EGF (EMD Millipore, catalog number: 01-102 ) (see Recipes)
  22. Rhodamine phalloidin (see Recipes)

Equipment

  1. Centrifuges
  2. Coverslips
  3. Tissue culture flask
  4. Parafilm
  5. Culture dish
  6. Humidified chamber
  7. Sharp forceps
  8. Water bath
  9. Culture hood

Procedure

Copyright: © 2012 The Authors; exclusive licensee Bio-protocol LLC.

Category

Cell Biology > Cell imaging > Fluorescence

Cell Biology > Cell movement > Cell migration

Biochemistry > Protein > Immunodetection > Immunostaining

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