Immunohistochemistry Protocol (Frozen) – Specific for Product: 4060

Specific For: Phospho-Akt (Ser473) (D9E) XP^® Rabbit mAb #4060

A. Solutions and Reagents

1. Xylene.
2. Ethanol (anhydrous denatured, histological grade 100% and 95%).
3. Hematoxylin (optional).
4. Fixative:
   1. Methanol.
   2. 16% formaldehyde.
      1. 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% H₂0₂ 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. SignalStain^® Antibody Diluent (#8112).
10. SignalStain^® Boost IHC Detection Reagent (HRP, Rabbit) (#8114)
11. 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

1. After sections have dried on the slide, fix as directed below:
   1. 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% H₂0₂ diluted in methanol.
3. Wash sections in wash buffer twice for 5 minutes.
4. Block each section with blocking solution for one hour at room temperature.
5. Remove blocking solution and add 100-400 µl diluted primary antibody to each section. (Dilute antibody in SignalStain^® Antibody Diluent #8112). Incubate overnight at 4°C.
   *Refer to product datasheet to determine the recommended dilution.
6. Remove antibody solution and wash sections three times with wash buffer for 5 minutes each.
7. Perform detection with SignalStain^® Boost IHC Detection Reagent (HRP, Rabbit) #8114. Cover sections with SignalStain^® Boost IHC Detection Reagent (HRP, Rabbit) #8114 and incubate in a humidified chamber for 30 minutes at room temperature.
8. Remove SignalStain^® Boost IHC Detection Reagent (HRP, Rabbit) #8114 and wash sections three times in wash buffer for 5 minutes each.
9. Add 100-400 µl DAB or suitable substrate to each section and monitor staining closely.
10. As soon as the sections develop, immerse slides in dH₂O.
11. If desired, counterstain sections in Hematoxylin per manufacturer’s instructions.
12. Wash sections in dH₂O two times for 5 minutes each.
13. 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.
14. Mount coverslips.