Cell Signaling Technology

Immunohistochemistry Protocol - Frozen for
SignalStain® Boost Detection Reagent

*IMPORTANT: See product datasheet for the appropriate antibody dilution, and fixative.

A. Solutions and Reagents

NOTE: Prepare solutions with Milli-Q or equivalently purified water.

  1. Xylene
  2. Ethanol (anhydrous denatured, histological grade 100% and 95%)
  3. Hematoxylin (optional)
  4. Fixative: For optimal fixative, please refer to the product datasheet.
    1. 10% neutral buffered formalin
    2. Acetone
    3. Methanol
    4. 3% formaldehyde: To prepare, add 18.75 ml 16% formaldehyde to 81.25 ml 1X PBS.
  5. 10X Tris Buffered Saline (TBS): To prepare 1L, add 24.2 g Trizma® base (C4H11NO3) and 80 g sodium chloride (NaCl) to 1L dH2O. Adjust pH to 7.6 with concentrated HCl.
  6. Wash buffer: 1X Tris Buffered Saline (TBS): To prepare 1L, add 100 ml 10X TBS to 900 ml dH2O.
  7. Methanol/Peroxidase: To prepare, add 10 mL 30% H2O2 to 90 ml methanol. Store at -20°C.
  8. Blocking Solution: 1X TBS/0.3% Triton-X 100/5% normal goat serum (#5425). To prepare, add 500 µl goat serum and 30 µl Triton-X 100 to 9.5 ml 1X TBS.
  9. Detection System: Use according to manufacturer’s recommendations. Available detection systems.*
  10. DAB Reagent or suitable substrate: Prepare according to manufacturer’s recommendations.

B. Sectioning

  1. For tissue stored at -80°C: Remove from freezer and equilibrate at -20°C for approximately 15 minutes 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 minutes before fixing (this helps sections adhere to slides).

C. Fixation

NOTE: Consult product datasheet to determine the optimal fixative.

  1. After sections have dried on the slide, fix in optimal fixative as directed below.
    1. 10% Neutral buffered formalin: 10 minutes at room temperature. Proceed with staining procedure immediately.
    2. Cold acetone: 10 minutes at -20°C. Air dry. Proceed with staining procedure immediately.
    3. Methanol: 10 minutes at -20°C. Proceed with staining procedure immediately.
    4. 3% Formaldehyde: 15 minutes at room temperature. Proceed with staining procedure immediately.
    5. 3% Formaldehyde/methanol: 15 minutes at room temperature in 3% formaldehyde, followed by 5 minutes in methanol at -20°C (do not rinse in between). Proceed with staining procedure immediately.

D. Staining

  1. Wash sections in wash buffer twice for 5 minutes.
  2. Incubate for 10 minutes at room temperature in 3% H2O2 diluted in methanol.
  3. Wash sections in wash buffer twice for 5 minutes.
  4. Block each section with 100–400 µl blocking solution for 1 hour at room temperature.
  5. Remove blocking solution and add 100–400 µl primary antibody diluted in blocking solution to each section. *
  6. Incubate overnight at 4°C.
  7. Equilibrate SignalStain® Boost Detection Reagent to room temperature.
  8. Remove antibody solution and wash sections in wash buffer three times for 5 minutes each.
  9. Cover section with 1–3 drops SignalStain® Boost Detection Reagent as needed. Incubate in a humidified chamber for 30 minutes at room temperature.
  10. Wash sections three times with wash buffer for 5 minutes each.
  11. Add 100–400 µl DAB or suitable substrate to each section and monitor staining closely.
  12. As soon as the sections develop, immerse slides in dH2O.
  13. If desired, counterstain sections in hematoxylin per manufacturer’s instructions.
  14. Wash sections in dH2O two times for 5 minutes each.
  15. 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.
  16. Mount coverslips.

*Available Detection Systems:

posted February 2010

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