Immunofluorescence Protocol with Methanol Fixation (IF Methanol-fixed)
IMPORTANT: Please refer to the APPLICATIONS section on the front page of the datasheet to determine if this product is validated and approved for use with this protocol.
A. Solutions and Reagents
NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalent grade water.
- 20X Phosphate Buffered Saline (PBS): To prepare 1 L 1X PBS: add 50 ml 20X PBS to 950 ml dH2O mix. Adjust pH to 8.0.
- Methanol, 100%
- Blocking Buffer (1X PBS / 5% normal serum / 0.3% Triton™ X-100): To prepare 10 ml, add 0.5 ml normal serum from the same species as the secondary antibody (e.g. Normal Goat Serum (#5425) to 9.5 ml 1X PBS) and mix well. While stirring, add 30 µl Triton™ X-100.
- Antibody Dilution Buffer (1X PBS / 1% BSA / 0.3% Triton™ X-100):
To prepare 10ml, add 30 µl Triton™ X-100 to 10 ml 1X PBS. Mix well then add 0.1g BSA (#9998), mix.
- Fluorochrome-conjugated secondary antibodies:
(Anti-mouse #4408, #4409, #8890, #4410) (Anti-rabbit #4412, #4413, #8889, #4414) (Anti-rat #4416, #4417, #4418).
- Prolong® Gold AntiFade Reagent (#9071), Prolong® Gold Antifade Reagent with DAPI (#8961).
B. Specimen Preparation - Cultured Cell Lines (IF-IC)
NOTE: Cells should be grown, treated, fixed and stained directly in multi-well plates, chamber slides or on coverslips.
- Aspirate liquid, then cover cells to a depth of 2–3 mm with ice-cold 100% methanol.
- Allow cells to fix for 15 minutes at -20°C.
- Aspirate fixative, rinse three times in PBS for 5 minutes each.
- Proceed with Immunostaining (Section C).
NOTE: All subsequent incubations should be carried out at room temperature unless otherwise noted in a humid light-tight box or covered dish/plate to prevent drying and fluorochrome fading.
- Block specimen in Blocking Buffer for 60 minutes.
- While blocking, prepare primary antibody by diluting as indicated on datasheet in Antibody Dilution Buffer.
- Aspirate blocking solution, apply diluted primary antibody.
- Incubate overnight at 4°C.
- Rinse three times in PBS for 5 minutes each.
NOTE: If using a fluorochrome-conjugated primary antibody, then skip to Section C, Step 8.
- Incubate specimen in fluorochrome-conjugated secondary antibody diluted in Antibody Dilution Buffer for 1–2 hours at room temperature in dark.
- Rinse in PBS as in step 5.
- Coverslip slides with Prolong® Gold Antifade Reagent (#9071) or Prolong® Gold Antifade Reagent with DAPI (#8961).
- For best results, examine specimens immediately using appropriate excitation wavelength. For long-term storage, store slides flat at 4°C protected from light.
posted December 2010
revised July 2016