The antibody was successfully validated for formalin-fixed paraffin-embedded human heart tissue using the following protocol:
1. Deparaffinize the paraffin-embedded sections by heating at 57°C for 60 minutes and rehydrate for 3 minutes in the following solutions: 2x xylene, 1x xylene:100% ethanol, 2x 100% ethanol, 1x 95% ethanol, 70% ethanol, 50% ethanol, and cold tap water.
2. Perform heat-induced epitope retrieval in 1 X citrate buffer (pH 6.0) and microwave at 750 W for 20 minutes.
3. Quench endogenous peroxidase activity with 0.1% H2O2 for 20 minutes.
4. Permeabilize with 0.05% saponin diluted in PBS for 5 minutes.
5. Use the Vectastain Elite ABC Universal Kit for immunohistochemical detection per the manufacturer's instructions.
6. Incubate sections with primary antibody anti-HHIP clone M01 WH0064399M1-100UG (1:100) at 4°C overnight. Omit primary antibodies for negative controls.
7. Visualize immunoreactivity with 3,3'-diaminobenzidine (DAB) substrate.
8. Counterstain cells for 30 seconds with hematoxylin and mount with Mowiol.
9. Examine staining under an inverted light microscope and digitally record at 40x magnification.