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Immunofluorescent Staining of Live Cells


Introduction

This protocol describes the procedure for direct immunofluorescent (IF) staining of live cells in culture using BioLite™ Antibodies. All steps should be performed in a sterile working environment using fresh culture medium.

 

Required Materials
  • BioLite Antibody
  • Cell Culture Medium
  • Pluripotent Stem Cells
Notes:
    • When using a primary antibody for the first time, it is recommended that the antibody be titrated for optimal performance specific to your cell type.
    • Cells can be grown, treated, and stained directly in multi-well plates.
    • Only open the antibody in a biological safety cabinet to prevent possible contamination.
    • Protect fluorescent conjugates from light.

Steps
  • 1. Refer to the Technical Data Sheet for the recommended starting concentration, and titrate the primary antibody for optimal performance on your cell type.
  • 2. Dilute the primary antibody in fresh cell culture medium to the determined optimum concentration.
  • 3. Aspirate the culture medium from the well of cells to be stained, and add the diluted antibody directly to the well of live cells.
  • 4. Incubate the cells in a 5% CO2 incubator at 37°C for 30 minutes.
  • 5. Aspirate the primary antibody and gently wash the cells twice with fresh culture medium.
  • 6. Add fresh cell culture medium to the well.
  • 7. Examine the cells using a fluorescence microscope and appropriate filters.
  • 8. Return cells to the incubator and continue to culture as normal.



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