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.
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:
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.
Actone fixation (ice cold) 10 minutes
Air dry 30 minutes in hood
Circle cytospin with a hydrophobic barrier pen.
Wash solution. Do not allow sections to dry for the remaining procedure.
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