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13079
Immunohistochemistry Application Solutions Kit (Rabbit)
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Immunohistochemistry Application Solutions Kit (Rabbit) #13079

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To Purchase # 13079S
Product # Size Price
13079S
1 Kit  (120 slides) $ 410

Reagents Not Included

  1. Xylene
  2. Ethanol anhydrous denatured, histological grade (100% and 95%)
  3. Deionized Water (dH2O)
  4. Antigen unmasking reagent
  5. 3% Hydrogen Peroxide
  6. Primary antibody
  7. Tris-Buffered Saline with Tween® 20 (TBST-10X, #9997)
  8. Hematoxylin (#14166) (optional)
  9. SignalStain® Mounting Medium (#14177)

Storage

All components in this kit are stable for at least 12 months past the reference date indicated on the component label when stored at 4°C and left unused.

Protocol

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Immunohistochemistry Application Solutions Kit (Rabbit) Protocol

NOTE: Please refer to the primary antibody product webpage for recommended primary antibody dilution buffer and recommended antibody dilution.

A Solutions and Reagents

SUPPLIED REAGENTS

  • Antibody Diluent: SignalStain® Antibody Diluent (#8112)
  • IHC Detection System: SignalStain® Boost IHC Detection Reagent (HRP, Rabbit): (#8114)
  • SignalStain® DAB Substrate Kit: (#8059)
    1. SignalStain® DAB Diluent (#11724)
    2. SignalStain® DAB Chromogen Concentrate (#11725)
  • Animal-Free Blocking Solution (5X): (#15019)

    1.0 ml of 1X Blocking Solution, add 200 μl of Animal-Free blocking solution and 800 μl of dH2O just before use. Always prepare fresh 1X solutions daily.

ADDITIONAL REAGENTS (NOT SUPPLIED)

  • Xylene
  • Ethanol, anhydrous denatured, histological grade (100% and 95%)
  • Deionized water (dH2O)
  • Antigen Unmasking:

    NOTE: Consult product datasheet for specific unmasking solution recommendation.

    1. Citrate: 10mM Sodium Citrate Buffer, pH 6.0: To prepare 1 L, add 2.94 g sodium citrate trisodium salt dihydrate (C6H5Na3O7•2H2O) to 1 L dH2O. Adjust pH to 6.0.
    2. EDTA: 1mM EDTA, pH 8.0: To prepare 1L, add 0.372 g EDTA (C10H14N2O8Na2•2H2O) to 1L dH2O. Adjust pH to 8.0.
    3. TE: 10mM Tris/1mM EDTA, pH 9.0: To prepare 1L, add 1.21 g Trizma® base (C4H11NO3) and 0.372 g EDTA (C10H14N2O8Na2) to 950 ml dH2O. Adjust pH to 9.0 and adjust final volume to 1L with dH2O.
    4. Pepsin: 1 mg/ml in Tris-HCL, pH 2.0.
  • 3% Hydrogen Peroxide: To prepare 100 ml, add 10 ml 30% H2O2 to 90 ml dH2O.
  • Primary Antibody
  • Wash Buffer: To prepare 1 L 1X TBST wash buffer: add 100 ml of 10X Tris Buffered Saline with Tween® 20 (TBST) (#9997) to 900 ml of dH2O, mix.
  • Hematoxylin #14166 (optional)
  • SignalStain® Mounting Medium #14177

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

NOTE: Consult product datasheet for specific unmasking solution recommendation.

  1. For Citrate: Bring slides to a boil in 10 mM sodium citrate buffer, pH 6.0 and 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 and follow with 15 min at a sub-boiling temperature. No cooling is necessary.
  3. For TE: Bring slides to a boil in 10 mM Tris/1 mM EDTA, pH 9.0 and maintain at a sub-boiling temperature for 18 min. Cool at room temperature for 30 min.
  4. For Pepsin: Digest for 10 min at 37 °C.

D. Staining

NOTE: Consult product datasheet for specific dilution recommendations for each primary antibody.

  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 100-150 μl blocking solution for 1 hr at room temperature.
  6. Remove blocking solution and add 100-200 μl primary antibody diluted in SignalStain® Antibody Diluent (#8112) to each section. Incubate overnight at 4°C.
  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 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 1 drop (30 μl) SignalStain® DAB Chromogen Concentrate to 1 ml SignalStain® DAB Diluent and mix well before use.
  12. Apply 100-400 μl SignalStain® DAB to each section and monitor closely. 1-10 min generally provides acceptable staining intensity.
  13. Immerse slides in dH2O.
  14. If desired, counterstain sections in 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 coverslips using SignalStain® Mounting Medium #14177
    1. Apply a small amount of SignalStain® Mounting Medium #14177 either on or near the section using a pipette. When the coverslip is applied, the medium will spread evenly across the coverslipped area.
    2. Allow the preparation to dry completely. Drying may take several hours when left on the lab bench, however this process can be accelerated by incubating the slides for 15-30 minutes at 60°C to 75°C. Please ensure the slides have cooled before storage.

posted June 2020

Protocol Id: 2105

Product Description

The Immunohistochemistry Application Solutions Kit (IHC-P) is designed to conveniently provide supporting reagents needed for immunohistochemistry staining in paraffin-embedded tissue samples or cell pellets (IHC-P). The reagents in this kit are thoroughly validated using our IHC-recommended rabbit polyclonal and monoclonal antibodies and will perform optimally with the CST immunohistochemistry staining protocol, ensuring accurate and reproducible results. This kit includes sufficient reagents for 120 slides based on a 100 µl assay volume. All reagents in this kit are available individually.

IMPORTANT: Please refer to the primary antibody data sheet to determine if the antibody is approved for use on paraffin-embedded samples (IHC-P) and for information regarding the appropriate antibody dilution, diluent, and antigen unmasking procedure.

For Research Use Only. Not For Use In Diagnostic Procedures.

Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.
SignalStain is a trademark of Cell Signaling Technology, Inc.
Tween is a registered trademark of ICI Americas, Inc.