IMPORTANT: Please refer to the APPLICATIONS section on the front page of product datasheet or product webpage 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). Please see product datasheet or product webpage for appropriate antibody dilution and unmasking solution. This protocol is recommended for both unconjugated and fluorophore conjugated antibodies.

NOTE: Some CST™ antibodies work optimally using an alternate protocol. Please see product datasheet for product-specific recommendations.

A. Solutions and Reagents

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

  1. 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.
  2. Formaldehyde: 16%, methanol free, Polysciences, Inc. (cat# 18814), use fresh and store opened vials at 4°C in dark. Dilute 1 in 4 in 1X PBS to make a 4% formaldehyde solution.
  3. 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 ml 1X PBS) and mix well. While stirring, add 30 µl Triton™ X-100.
  4. 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.
  5. Fluorochrome-conjugated Secondary Antibodies: (Anti-mouse #4408, #4409, #8890, #4410) (Anti-rabbit #4412, #4413, #8889, #4414) (Anti-rat #4416, #4417, #4418).
  6. Prolong® Gold Antifade Reagent (#9071), Prolong® Gold Antifade Reagent with DAPI (#8961).

Reagents specific to IF-P application:

  1. Xylene.
  2. Ethanol, anhydrous denatured, histological grade, 100% and 95%.
  3. Antigen Unmasking:
    1. For Citrate: 10 mM Sodium Citrate Buffer: To prepare 1 L add 2.94 g (C6H5Na3O7•2H2O) to 1 L dH2O. Adjust pH to 6.0.
    2. For 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 liquid, then cover cells to a depth of 2–3 mm with 4% formaldehyde diluted in warm PBS.

    NOTE: Formaldehyde is toxic, use only in a fume hood.

  2. Allow cells to fix for 15 min at room temperature.
  3. Aspirate fixative, rinse three times in 1X PBS for 5 min 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. Wash three times in xylene for 5 min each.
    2. Wash two times in 100% ethanol for 10 min each.
    3. Wash two times in 95% ethanol for 10 min each.
    4. Rinse sections two times in dH2O for 5 min each.
  2. Antigen Unmasking: NOTE: Consult product datasheet or product webpage 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 min. Cool slides on bench top for 30 min.
    2. For EDTA: Bring slides to a boil in 1 mM EDTA pH 8.0 followed by 15 min 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, fix immediately, as follows:
    1. Cover sections with 4% formaldehyde diluted in warm 1X PBS.
    2. Allow sections to fix for 15 min at room temperature.
    3. Rinse slides three times in PBS for 5 min 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. Block specimen in blocking buffer for 60 min.
  2. While blocking, prepare primary antibody by diluting as indicated on datasheet in antibody dilution buffer.
  3. Aspirate blocking solution, apply diluted primary antibody.
  4. Incubate overnight at 4°C.
  5. Rinse three times in 1X PBS for 5 min each.

    NOTE: If using a fluorochrome-conjugated primary antibody, then skip to Section C, Step 8.

  6. Incubate specimen in fluorochrome-conjugated secondary antibody diluted in antibody dilution buffer for 1–2 hr at room temperature in the dark.
  7. Rinse three times in 1X PBS for 5 min each.
  8. Coverslip slides with Prolong® Gold Antifade Reagent (#9071) or Prolong® Gold Antifade Reagent with DAPI (#8961).
  9. For best results, allow mountant to cure overnight at room temperature. For long-term storage, store slides flat at 4°C protected from light.

posted November 2006

revised November 2013

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