A. Solutions and Reagents

  1. Xylene.
  2. Ethanol, anhydrous denatured, histological grade (100% and 95%).
  3. Deionized water (dH2O).
  4. Hematoxylin (optional).
  5. Wash Buffer.

    NOTE: Additional 10X TBST will be required for washes.

    1. Tris Buffered Saline with Tween® 20 (TBST-10X) #9997: To prepare wash buffer add 100 μl of Tris Buffered Saline with Tween® 20 (TBST-10X) #9997 to 900 ml of dH2O. OR
    2. 10X Tris Buffered Saline (TBS): To prepare 1 L, add 24.2 g Trizma® base (C4H11NO3) and 80 g sodium chloride (NaCl) to 1 L dH2O. Adjust pH to 7.6 with concentrated HCl. 1X TBS/0.1% Tween® 20 (1X TBST): To prepare 1 L, add 100 ml 10X TBS to 900 ml dH2O. Add 1 ml Tween® 20 and mix.
  6. Antibody Diluent: SignalStain® Antibody Diluent #8112
  7. Antigen Unmasking: 10 mM Sodium Citrate Buffer: To prepare 1 L, add 2.94 g sodium citrate trisodium salt dihydrate (C6H5Na3O72H2O) to 1 L dH2O. Adjust pH to 6.0.
  8. 3% Hydrogen Peroxide: To prepare, add 10 ml 30% H2O2 to 90 ml dH2O.
  9. Blocking Solution: 1X TBST/5% normal goat serum: Add 100 μl 10X TBST (#9997) and 50 μl normal goat serum (#5425) to 850 μl dH2O.
  10. Detection System: SignalStain® Boost IHC Detection Reagent (HRP, Rabbit) (#8114).
  11. Substrate: SignalStain® DAB Substrate Kit (#8059), which includes SignalStain® DAB Diluent (#11724) and SignalStain® DAB Chromogen Concentrate (#11725).

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 min each.
    2. Incubate sections in two washes of 100% ethanol for 10 min each.
    3. Incubate sections in two washes of 95% ethanol for 10 min each.
  2. Wash sections twice in dH2O for 5 min each.

C. Antigen Unmasking

  1. 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.

D. Staining

  1. Wash sections in dH2O three times for 5 min each.
  2. Incubate sections in 3% hydrogen peroxide for 10 min.
  3. Wash sections in dH2O twice for 5 min each.
  4. Wash section in wash buffer for 5 min.
  5. Block each section with 100–200 μl blocking solution for 1 hr at room temperature.
  6. Remove blocking solution and add 100–200 μl primary antibody diluted at 1:500 in SignalStain® Antibody Diluent (#8112) to each section. Incubate overnight at 4°C.
    1. Rabbit (DA1E) mAb XP® SignalStain® Isotype Control (#3900) is used as a negative control. Dilute at 1:500 in SignalStain® Antibody Diluent (#8112) and apply 100–200 μl to each section.
  7. Equilibrate SignalStain® Boost Detection Reagent (#8114) to room temperature.
  8. Remove antibody solution and wash sections in wash buffer three times for 5 min each.
  9. Cover section with 1–3 drops SignalStain® Boost Detection Reagent (#8114) as needed. Incubate in a humidified chamber for 30 min at room temperature.
  10. Wash sections three times with wash buffer for 5 min each.
  11. Add 30 μl SignalStain® DAB Chromogen Concentrate (#11725) to 1 ml SignalStain® DAB Diluent (#11724) and mix well before use.
  12. Apply 100–400 μl SignalStain® DAB to each section and monitor closely. 1–10 minutes generally provides an acceptable staining intensity.
  13. Immerse slides in dH2O.
  14. If desired, counterstain sections in hematoxylin per manufacturer’s instructions.
  15. Wash sections in dH2O two times for 5 min each.
  16. Dehydrate sections:
    1. Incubate sections in 95% ethanol two times for 10 sec each.
    2. Repeat in 100% ethanol, incubating sections two times for 10 sec each.
    3. Repeat in xylene, incubating sections two times for 10 sec each.
  17. Mount coverslips.

posted July 2013

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