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:

• SignalStain^® Boost IHC Detection Reagent (HRP, Rabbit) #8114
• SignalStain^® Boost IHC Detection Reagent (HRP, Mouse) #8125