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Immunohistochemistry (Paraffin)

A. Solutions and Reagents

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

  1. Xylene.
  2. Ethanol, anhydrous denatured, histological grade (100% and 95%).
  3. Deionized water (dH2O).
  4. Wash Buffer:
    1. 1X Tris Buffered Saline with Tween® 20 (TBST): To prepare 1 L 1X TBST, add 100 ml 10X Tris Buffered Saline with Tween® 20 (TBST)(#9997) to 900 ml dH20, mix.
    2. 1X Phosphate Buffered Saline (PBS): To prepare 1L 1X PBS, add 100 mL 10X Wash Buffer, Phosphate Buffered Saline(#12528) to 900 ml dH20, mix.
  5. SignalStain® Antibody Diluent: (#8112)
  6. 1X EDTA Unmasking Solution: To prepare 250 mL of 1X EDTA unmasking solution, dilute 25 ml of SignalStain® EDTA Unmasking Solution (10X) (#14747) with 225 ml of dH2O.
  7. 3% Hydrogen Peroxide: To prepare, add 10 ml 30% H2O2 to 90 ml dH2O.
  8. Blocking Solution: 1X TBST/5% Normal Goat Serum or 1X Animal-Free Blocking Solution.
    1. TBST/5% Normal Goat Serum: to 5 ml 1X TBST, add 250 µl Normal Goat Serum (#5425).
    2. 1X Animal-Free Blocking Solution: to 4 mL of dH2O, add 1 ml of Animal-Free Blocking Solution (5X) (#15019).
  9. Prolong® Gold AntiFade Reagent(#9071), Prolong® Gold AntiFade Reagent with DAPI(#8961).
  10. (optional) TrueBlack® Lipofuscin Autofluorescence Quencher (#92401).

B. Deparaffinization/Rehydration

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

  1. Deparaffinize/hydrate sections:
    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.
  2. Wash sections twice in dH2O for 5 minutes each.

C. Antigen Unmasking

  1. Heat slides submersed in 1X EDTA unmasking solution in a microwave until boiling is initiated; follow with 15 min at sub-boiling temperature (95°-98°). No cooling is necessary.

    2. D. Staining

    1. Wash sections in dH2O three times for 5 minutes each.
    2. Incubate sections in 1X TBST for 5 min.
    3. Block each section with 100-400 µl of preferred blocking solution for 1 hour at room temperature.
    4. Remove blocking solution and add 100-400 µl primary antibody diluted in SignalStain® Antibody Diluent to each section.
    5. Incubate overnight at 4°C.
    6. Rinse three times in 1X PBS for 5 min each protected from light.
      NOTE: See below for optional TrueBlack® Lipofuscin Autofluorescence Quencher protocol.
    7. Coverslip slides with Prolong® Gold Antifade Reagent or Prolong® Gold Antifade Reagent with DAPI.
    8. For best results, allow mountant to cure overnight at room temperature. For long-term storage, store slides flat at 4°C protected from light.

    TrueBlack® Lipofuscin Autofluorescence Quencher protocol

    Following Section D Step 6:

    IMPORTANT: TrueBlack® Lipofuscin Autofluorescence Quencher is not compatible with detergent. Any steps involving detergent must be done before applying TrueBlack® Lipofuscin Autofluorescence Quencher.

    1. Prepare TrueBlack® Lipofuscin Autofluorescence Quencher solution by diluting 1:20 in 70% ethanol. Vortex to mix.
      NOTE: Quenching solution should be made fresh prior to use and discarded if precipitate is visible. We recommend heating the vial of stock solution of TrueBlack® Lipofuscin Autofluorescence Quencher, 20X in DMF to 70°C prior to dilution in order to avoid precipitate formation.
    2. Immediately cover tissue sections with 100 µL - 200 µL of quenching solution for 30 seconds at room temperature.
      IMPORTANT: Do not allow sections to dry out. Sections may tolerate longer incubations (up to 3 minutes) so long as they remain hydrated.
    3. Tap slides on an absorbent towel to collect excess TrueBlack® Lipofuscin Autofluorescence Quencher before transferring to 1X PBS.
    4. Rinse three times in 1X PBS for 5 min each.
    5. Proceed with counterstaining/mounting.