Immunofluorescence Protocol for use with PathScan^® Multiplex IF Kits

IMPORTANT: Please refer to the APPLICATIONS section on the front page of the datasheet to determine if this kit has been 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 warm PBS for use.
• 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 goat 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 25 ml, add 2.5 ml 10X PBS to 22.5 ml dH₂O, mix. Add 0.25 g BSA and mix well. While stirring, add 75 µl Triton X-100.
• 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 multi-well plates, chamber slides, or on coverslips.

1. Aspirate culture medium, and then cover cells to a depth of 2–3 mm with 4% formaldehyde diluted in 1X PBS warmed to 37°C.
   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 procedure.

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.

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.

III. Frozen/Cryostat Sections (IF-F)

NOTE: Fresh frozen/unfixed sections should be fixed immediately in 4% formaldehyde as follows to preserve signaling epitopes.

1. Cover sections with 4% formaldehyde diluted in 1X PBS warmed to 37°C.
   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.

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. Block specimen in Blocking Buffer for 60 minutes.
2. While blocking, prepare primary cocktail by diluting as indicated on datasheet in Antibody Dilution Buffer.
3. Aspirate blocking solution, apply diluted primary cocktail.
4. Incubate overnight at 4°C.
5. Rinse three times in PBS for 5 minutes each.
6. Prepare detection cocktail by diluting as indicated on datasheet in Antibody Dilution Buffer.
7. Incubate 1–2 hours at room temperature in the dark.
8. Rinse three times in PBS for 5 minutes each.
9. Coverslip slides with Prolong^® Gold Antifade Reagent.
10. For best results examine specimens immediately using appropriate excitation wavelengths. For long-term storage, store slides at 4°C protected from light.