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Immunohistochemistry (Frozen)

#2976 Phospho-mTOR (Ser2448) (49F9) Rabbit mAb (IHC Specific)用プロトコール

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. Fixative: Acetone.
  5. Wash Buffer: 1X Tris Buffered Saline (TBS).

    To prepare 1L 1X TBS add 100 ml 10X Tris Buffered Saline (#12498) to 900 ml dH2O, mix.

  6. 3% Hydrogen Peroxide: To prepare 100 ml, add 10 ml 30% H2O2 to 90 ml dH2O.
  7. Blocking Solution: 1X TBS/0.3% Triton X-100/5% Normal Goat Serum or 1X Animal-Free Blocking Solution.
    1. 1X TBS/0.3% Triton X-100/5% Normal Goat Serum: add 500 µl Normal Goat Serum (#5425) and 30 µl Triton X-100 to 9.5 ml 1X TBS.
    2. 1X Animal-Free Blocking Solution: to 4 mL of dH2O, add 1 ml of Animal-Free Blocking Solution (5X) (#15019).
  8. Antibody Diluent 1X TBS/0.3% Triton™ X-100/5% Normal Goat Serum: add 500 µl Normal Goat Serum (#5425) and 30 µl Triton™ X-100 to 9.5 ml 1X TBS.
  9. Detection System: SignalStain® Boost IHC Detection Reagents (HRP, Rabbit #8114).
  10. Substrate: Vector® NovaRED™ (Vector Laboratories).
  11. Hematoxylin: Hematoxylin (#14166).
  12. Mounting Medium: SignalStain® Mounting Medium (#14177).

B. Sectioning

  1. For tissue stored at -80°C: Remove from freezer and equilibrate at -20°C for approximately 15 min before attempting to section. This may prevent cracking of the block when sectioning.
  2. Section tissue at a range of 6–8 µm and place on positively charged slides.
  3. Allow sections to air dry on bench for a few min before fixing (this helps sections adhere to slides).

C. Fixation

  1. Fix sections in cold acetone for 10 min at -20°C. Air dry. Proceed with staining procedure immediately (Section D).

D. Staining

  1. Wash sections in wash buffer two times for 5 min.
  2. Incubate for 10 min at room temperature in 3% Hydrogen Peroxide.
  3. Wash sections in wash buffer two times for 5 min.
  4. Block each section with 100–400 µl of preferred blocking solution for 1 hr at room temperature.
  5. Remove blocking solution and add 100–400 µl primary antibody diluted in recommended antibody diluent to each section.
  6. Incubate overnight at 4°C.
  7. Equilibrate SignalStain® Boost Detection Reagent (HRP, Rabbit #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 (HRP, Rabbit #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. Prepare Vector® NovaRED™ (Vector Laboratories) per manufacturer's instructions.
  12. Apply 100-400 µl substrate to each section and monitor staining closely. 1-10 minutes generally provides an acceptable staining intensity.
  13. Immerse slides in dH2O.
  14. If desired, counterstain sections with hematoxylin (#14166).
  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 sections with coverslips and mounting medium (#14177).

posted February 2010

revised March 2016

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