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  4. Immunohistochemistry Protocol - Frozen for SignalStain® Boost Detection Reagent

Immunohistochemistry Protocol - Frozen for SignalStain® Boost Detection Reagent

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. Hematoxylin (#14166).
  4. 20X Phosphate Buffered Saline (PBS): (#9808) To prepare 1 L 1X PBS: add 50 ml 20X PBS to 950 ml dH2O, mix.
  5. Fixative Options:
    1. 10% neutral buffered formalin.
    2. Acetone.
    3. Methanol.
    4. 3% formaldehyde: To prepare 100 ml, add 18.75 ml 16% formaldehyde to 81.25 ml 1X PBS.
  6. 10X Tris Buffered Saline (TBS) Wash Buffer: (#12498) To prepare 1 L 1X TBS add 100 ml of 10X TBS to 900 ml dH2O, mix.
  7. Methanol/Peroxidase: To prepare, add 10 ml 30% H2O2 to 90 ml methanol. Store at -20°C.
  8. Blocking Solution: 1X TBST/5% Normal Goat Serum or 1X Animal-Free Blocking Solution.
    1. 1X TBST/5% Normal Goat Serum: add 250 µL Normal Goat Serum (#5425) to 5 mL 1X TBST.
    2. 1X Animal-Free Blocking Solution: to 4 mL of dH2O, add 1 mL of Animal-Free Blocking Solution (5X) (#15019).
  9. Detection System: SignalStain® Boost IHC Detection Reagents (HRP, Mouse #8125; HRP, Rabbit #8114; HRP, Rat #72838; HRP, Goat #63707; AP, Mouse #31926; AP, Rabbit #18653; AP, Rat #15764; AP, Goat #26927).
  10. Substrate: HRP compatible: SignalStain® DAB Substrate Kit (#8059); SignalStain® Vivid Purple Peroxidase Substrate Kit (#96632); SignalStain® Deep Black Peroxidase Substrate Kit (#72986); SignalStain® Radiant Yellow Peroxidase Substrate Kit (#69644); AP compatible: SignalStain® Vibrant Red Alkaline Phosphatase Substrate Kit (#76713); SignalStain® Ultra Blue Alkaline Phosphatase Substrate Kit (#12824).
  11. Mounting Medium: SignalStain® Mounting Medium II (#84583); SignalStain® Aqueous Mounting Medium (#27290).

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 Options

NOTE: The optimal fixative must be determined for each antibody.

After sections have dried on the slide, fix in optimal fixative as directed below.

  1. 10% Neutral buffered formalin: 10 min at room temperature. Proceed with staining procedure immediately (Section D).
  2. Cold acetone: 10 min at -20°C. Air dry. Proceed with staining immediately (Section D).
  3. Methanol: 10 min at -20°C. Proceed with staining immediately (Section D).
  4. 3% Formaldehyde: 15 min at room temperature. Proceed with staining immediately (Section D).
  5. 3% Formaldehyde/methanol: 15 min at room temperature in 3% formaldehyde, followed by 5 min in methanol at -20°C (do not rinse in between). Proceed with staining 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 methanol/peroxidase.
  3. Wash sections in wash buffer two times for 5 min.
  4. Block each section with 100-400 µl 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 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.
  10. Incubate in a humidified chamber for 30 min at room temperature.
  11. Wash sections three times with wash buffer for 5 min each.
  12. Prepare the substrate as recommended in the Product Usage Information.
  13. Apply 100-400 µL substrate to the slides. Monitor the reaction. The recommended reaction time differs depending upon the substrate used. Refer to the Product Usage Information for specific guidance.
  14. Immerse slides in dH2O.
  15. If desired, counterstain sections with hematoxylin per manufacturer's instructions.
  16. Wash sections in dH2O two times for 5 min each.
  17. 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.
  18. Mount sections with coverslips.