Immunofluorescence Protocol with Methanol Permeabilization (IF Methanol-perm)

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 on cultured cell lines (IF-IC), paraffin-embedded samples (IF-P), or frozen tissue sections (IF-F).

A. Solutions and Reagents

NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalent grade water.

• 20X Phosphate Buffered Saline (PBS) (#9808): To prepare 1 L 1X PBS: add 50 ml 20X PBS to 950 ml dH₂O, mix. NOTE: Adjust pH to 8.0.
• Formaldehyde, 16%, methanol free, Polysciences, Inc. (cat# 18814), use fresh, store opened vials at 4°C in dark, dilute in PBS for use. Dilute 1 in 4 in 1X PBS to make a 4% formaldehyde solution.
• 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 10 ml, 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

I. Cultured Cell Lines (IF-IC)

NOTE: Cells should be grown, treated, fixed and stained directly in multiwell plates, chamber slides or on coverslips.

1. Aspirate liquid, then cover cells to a depth of 2–3 mm with 4% formaldehyde in PBS.
   NOTE: Formaldehyde is toxic, use only in fume hood.
2. Allow cells to fix for 15 minutes at room temperature.
3. Aspirate fixative, rinse three times in PBS for 5 minutes each.
4. Proceed with Immunostaining (Section C).

II. Frozen/Cryostat Sections (IF-F)

1. For fixed frozen tissue proceed with Immunostaining (Section C).
2. For fresh, unfixed frozen tissue, please fix immediately, as follows:
   1. Cover sections with 4% formaldehyde in PBS.
      NOTE: Formaldehyde is toxic, use only in fume hood.
   2. Allow sections to fix for 15 minutes at room temperature.
   3. Rinse slides three times in PBS for 5 minutes each.
   4. Proceed with Immunostaining (Section C).

C. Immunostaining

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.

1. Methanol Permeabilization Step: Cover cells or tissue sections with ice-cold 100% methanol (use enough to cover completely to a depth of 3–5 mm, DO NOT LET DRY), incubate in methanol for 10 minutes at –20°C, rinse in PBS for 5 minutes.
2. Block specimen in Blocking Buffer for 60 minutes.
3. While blocking, prepare primary antibody by diluting as indicated on datasheet in Antibody Dilution Buffer.
4. Aspirate blocking solution, apply diluted primary antibody.
5. Incubate overnight at 4°C.
6. Rinse three times in PBS for 5 minutes each.
   NOTE: If using a fluorochrome-conjugated primary antibody, then skip to Section C, Step 8.
7. Incubate specimen in fluorochrome-conjugated secondary antibody diluted in Antibody Dilution Buffer for 1–2 hours at room temperature in dark.
8. Rinse in PBS as in step 6.
9. Coverslip slides with Prolong^® Gold Antifade Reagent (#9071) or Prolong^® Gold Antifade Reagent with DAPI (#8961).
10. For best results examine specimens immediately using appropriate excitation wavelength. For long term storage, store slides flat at 4°C protected from light.