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Immunohistochemistry (Paraffin)

#2197 Phospho-Chk2 (Thr68) (C13C1) Rabbit mAb用プロトコール

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:
    1. 1X Tris Buffered Saline with Tween® 20 (TBST): To prepare 1L 1X TBST add 100 ml 10X Tris Buffered Saline with Tween® 20 (#9997) to 900 ml dH20, mix.
  6. SignalStain® Antibody Diluent (#8112).
  7. 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.
  8. 3% Hydrogen Peroxide: To prepare, add 10 ml 30% H2O2 to 90 ml dH2O.
  9. Blocking Solution: TBST/5% normal goat serum (#5425): to 5 ml 1X TBST add 250 µl normal goat serum.
  10. Detection System: VECTASTAIN® Elite ABC, including biotinylated secondary antibody (Vector Laboratories).
  11. Substrate: SignalStain® DAB Substrate Kit (#8059).

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

For Citrate: Bring slides to a boil in 10 mM sodium citrate buffer, pH 6.0; 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 minutes each.
  2. Incubate sections in 3% hydrogen peroxide for 10 minutes.
  3. Wash sections in dH2O twice for 5 minutes each.
  4. Wash sections in wash buffer for 5 minutes.
  5. Block each section with 100-400 µl blocking solution for 1 hour at room temperature.
  6. Remove blocking solution and add 100-400 µl primary antibody diluted in SignalStain® Antibody Diluent (#8112) to each section. Incubate overnight at 4°C.
  7. Prepare ABC solution per manufacturer's recommendations.
  8. Remove primary antibody and wash section three times with wash buffer for 5 minutes each.
  9. Add 100-400 µl biotinylated secondary antibody, diluted in TBST per manufacturer’s recommendation, to each section. Incubate 30 minutes at room temperature.
  10. Remove secondary antibody solution and wash sections three times with wash buffer for 5 minutes each.
  11. Cover sections with 100-400 µl pre-mixed ABC solution as needed and incubate in a humidified chamber for 30 min at room temperature.
  12. Wash section three times with wash buffer for 5 min each.
  13. Add 1 drop (30 µl) SignalStain® DAB Chromogen Concentrate to 1 ml SignalStain® DAB Diluent and mix well before use.
  14. Apply 100-400 µl SignalStain® DAB to each section and monitor closely. 1-10 minutes generally provides an acceptable staining intensity.
  15. Immerse slides in dH20.
  16. If desired, counterstain sections in hematoxylin per manufacturer’s instructions.
  17. Wash sections in dH2O two times for 5 minutes each.
  18. Dehydrate sections:
    1. Incubate sections in 95% ethanol two times for 10 seconds each.
    2. Repeat in 100% ethanol, incubating sections two times for 10 seconds each.
    3. Repeat in xylene, incubating sections two times for 10 seconds each.
  19. Mount coverslips.

posted April 2015

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