Tissue Microarray Immunohistochemistry for ATP1A1

Paraffin-embedded tissue microarray (TMA) slides, BR1201, were purchased from US Biomax (Rockville, MD, USA). The slides were de-paraffinized, followed by antigen retrieval in sodium citrate buffer, pH 6.8 (ScyTek Laboratories, Logan, UT, USA), for 20 min in a microwave oven. TMA specimens were probed with an anti-ATP1A1 antibody (Abcam, USA) using the VECTASTAIN ABC kit (Vector Labs, Newmark, CA, USA), with staining visualized with a NovaRED substrate (Vector Labs), followed by counterstaining with hematoxylin. The stained slides were subsequently scanned using a ZEISS Axio Scan Z1 microscope (Carl Zeiss Microscopy, Oberkochen, Germany), with staining intensity calculated by the IHC profiler [27 (link)] plug-in in Image J software.