Immunofluorescence Protocol with Methanol Permeabilization

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 Milli-Q or equivalently purified water.

• 10X Phosphate Buffered Saline (PBS): To prepare 1 L add 80 g sodium chloride (NaCl), 2 g potassium chloride (KCl), 14.4 g sodium phosphate, dibasic (Na₂HPO₄) and 2.4 g potassium phosphate, monobasic (KH₂PO₄) to 1 L dH₂O. 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.
• Methanol, 100%
• Blocking Buffer (1X PBS / 5% normal goat serum (#5425) / 0.3% Triton X-100):
  To prepare 25 ml, add 2.5 ml 10X PBS, 1.25 ml normal serum from the same species as the secondary antibody* (e.g., normal goat serum, normal donkey serum) and 21.25 ml dH₂O and mix well. While stirring, add 75 µl Triton X-100.
• Antibody Dilution Buffer (1X PBS / 1% BSA / 0.3% Triton X-100):
  To prepare 40 ml, add 4 ml 10X PBS to 36 ml dH₂O, mix. Add 0.4 g BSA and mix well. While stirring, add 120 µl Triton X-100.
• Fluorochrome-conjugated secondary antibody (recommended secondary antibodies)
  NOTE: When using any primary or fluorochrome-conjugated secondary antibody for the first time, titrate the antibody to determine which dilution allows for the strongest specific signal with the least background for your sample.
• Prolong^® Gold Antifade Reagent (Invitrogen, Eugene, OR, cat# P36930)

Reagents specific to IF-P application:

• Xylene
• Ethanol, anhydrous denatured, histological grade, 100% and 95%
• Antigen Unmasking:
  • Citrate: 10 mM Sodium Citrate Buffer: To prepare 1 L add 2.94 g sodium citrate trisodium salt dihydrate (C₆H₅Na₃O₇•2H₂O) to 1 L dH₂O. Adjust pH to 6.0.
  • EDTA: 1 mM EDTA: To prepare 1 L add 0.372 g EDTA (C₁₀H₁₄N₂O₈Na₂•2H₂O) to 1 L dH₂O. Adjust pH to 8.0.

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. Paraffin Sections (IF-P)

NOTE: Do not allow slides to dry at any time during this process.

1. Deparaffinization/Rehydration:
   1. Incubate sections in three washes of xylene for 5 minutes each.
   2. Incubate sections in two washes of 100% ethanol for 10 minutes each.
   3. Incubate sections in two washes of 95% ethanol for 10 minutes each.
   4. Rinse sections twice in dH₂O for 5 minutes each.
2. Antigen Unmasking:
   NOTE: Consult product datasheet for specific recommendation for the unmasking solution.
   1. For Citrate: Bring slides to a boil in 10 mM sodium citrate buffer pH 6.0, then maintain at a sub-boiling temperature for 10 minutes. Cool slides on bench top for 30 minutes.
   2. For EDTA: Bring slides to a boil in 1 mM EDTA pH 8.0 followed by 15 minutes at a sub-boiling temperature. No cooling is necessary.
3. Proceed with Immunostaining (Section C).

III. 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 2–4% 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 primary antibodies directly conjugated with Alexa Fluor^® fluorochromes, then skip to step C8.
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.
10. For best results examine specimens immediately using appropriate excitation wavelength. For long term storage, store slides flat at 4°C protected from light.

*Recommended Secondary Antibodies:

Anti-Rabbit

• Anti-Rabbit IgG (H+L), F(ab')₂ Fragment (Alexa Fluor^® 488 Conjugate) #4412
• Anti-Rabbit IgG (H+L), F(ab')₂ Fragment (Alexa Fluor^® 555 Conjugate) #4413
• Anti-Rabbit IgG (H+L), F(ab')₂ Fragment (Alexa Fluor^® 647 Conjugate) #4414

Anti-Mouse

• Anti-Mouse IgG (H+L), F(ab')₂ Fragment (Alexa Fluor^® 488 Conjugate) #4408
• Anti-Mouse IgG (H+L), F(ab')₂ Fragment (Alexa Fluor^® 555 Conjugate) #4409
• Anti-Mouse IgG (H+L), F(ab')₂ Fragment (Alexa Fluor^® 647 Conjugate) #4410

Anti-Rat

• Anti-Rat IgG (H+L), (Alexa Fluor^® 488 Conjugate) #4416
• Anti-Rat IgG (H+L), (Alexa Fluor^® 555 Conjugate) #4417
• Anti-Rat IgG (H+L), (Alexa Fluor^® 647 Conjugate) #4418