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2-Step Immunofluorescence Protocol: Cells Grown in Culture and Cytospin Preparations

See the 2-step immunofluorescence protocol to be used for cells grown in culture and cytospin preparations. Includes required reagents, preparation steps and procedure.

For antibodies made in Rabbits

Materials:

  • Acetone
  • Hydrophobic barrier pen
  • Wash Solution
  • Normal Goat Serum Blocking Solution
  • IHC Antibody Diluent
  • Goat anti-Rabbit IgG Antibody-DyLight conjugated (Bethyl cat# A120-101D2, A120-101D3, A120-101D4, or cross-adsorbed antibodies A120-201D2, A120-201D3, A120-201D4)
  • Fluorescent mounting media with or without DAPI
  • Coverglass

Preparation of Materials:

Normal Goat Serum Blocking Solution

20 ml normal goat serum (Filter sterilize with 0.2 µM filter)
Volume to 100 ml with PBS or TBS.
Store 2-8 °C, discard after 3 months.


IHC Antibody Diluent (50mM TBS, 1% BSA)

200ml 1X TBS
2 g BSA (Sigma A9647)
Volume to 100 ml with PBS or TBS.
Store 2-8 °C, discard after 1 year.

*May want to use a perservative.


Wash solution

Can be any of the following: PBS, TBS or 0.01% Tween 20 in dH20. TBS wash solution is recommended for phospho antibodies.


Secondary Antibody

Goat anti-Rabbit IgG Antibody-Dylight conjugated. (Bethyl cat# A120-101D2, A120-101D3, A120-101D4, or cross-adsorbed antibodies A120-201D2, A120-201D3, A120-201D4) Dilute 1:100 with IHC antibody diluent. Prepare just prior to use. Protect from light. Optimal working dilutions should be determined experimentally by the investigator.


Procedure:

  1. For cells in chambered microscope slides or cells grown on coverslips: Gently rinse off media with PBS 3 changes for 1 minute each. For cytospins: Allow to air dry after preparation.
  2. Actone fixation (ice cold) 10 minutes
  3. Air dry 30 minutes in hood
  4. Circle cytospin with a hydrophobic barrier pen.
  5. Wash solution. Do not allow sections to dry for the remaining procedure.
  6. Normal Goat Serum Blocking Solution - 15 minutes
  7. Primary Antibody Incubation: 30 minutes room temperature.
    • Prepare primary antibody with IHC Antibody Diluent.
    • Optimal working dilutions should be determined experimentally by the investigator.
    • Refer to Bethyl antibody datasheet for suggested dilution range.
  8. Wash solution- 3 changes for 5 minutes each
  9. Secondary Antibody Incubation: 30 minutes - room temperature. Protect from light.
  10. Wash Solution - 3 changes for 5 minutes each
  11. Remove chamber from microscope slide
  12. Mount with fluorescent mounting media and coverslip. Use fluorescent mounting media with DAPI if counterstaining is desired.
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