Immunohistochemistry Protocol - Frozen for
SignalStain^® Boost Detection Reagent

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

A. Solutions and Reagents

NOTE: Prepare solutions with 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 1 L, add 24.2 g Trizma^® base (C₄H₁₁NO₃) and 80 g sodium chloride (NaCl) to 1 L dH₂O. Adjust pH to 7.6 with concentrated HCl.
6. Wash buffer: 1X Tris Buffered Saline (TBS): To prepare 1 L, 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: SignalStain^® Boost IHC Detection Reagents (mouse #8125, rabbit #8114).
10. Substrate: SignalStain^® DAB Substrate Kit (#8059).

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

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 min at room temperature. Proceed with staining procedure immediately.
   2. Cold acetone: 10 min at −20°C. Air dry. Proceed with staining procedure immediately.
   3. Methanol: 10 min at −20°C. Proceed with staining procedure immediately.
   4. 3% Formaldehyde: 15 min at room temperature. Proceed with staining procedure immediately.
   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 procedure immediately.

D. Staining

1. Wash sections in wash buffer twice for 5 min.
2. Incubate for 10 min at room temperature in 3% H₂O₂ diluted in methanol.
3. Wash sections in wash buffer twice 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 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 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 minutes generally provides an acceptable staining intensity.
13. Immerse slides in dH₂O.
14. If desired, counterstain sections in hematoxylin per manufacturer’s instructions.
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.