Immunohistochemical staining of tissue arrays

COC1021 tissue arrays (Pantomics, Inc.), which have defined clinical diagnosis and clinicopathological information, were used for immunohistochemical staining (49 (link)). Firstly, tissue arrays with 4 µm core thickness were deparaffinated and hydrated using routine protocols. Antigen retrieval was performed by steaming in Tris-EDTA buffer (pH 9.0) for 20 min, and arrays were then blocked in 1.5% (v/v) Normal Horse Serum Blocking Solution (Vector Laboratories, Inc.) for 2 h at room temperature. The specific target protein was immunodetected using an adequate primary antibody concentration [ubiquitin-conjugating enzyme E2 N (UBE2N): anti-Ube2N, cat. no. ab117090, dilution 1:20, Abcam; inosine monophosphate dehydrogenase 1 (IMPDH1): anti-IMPDH1, cat. no. ab84957, 1:20, Abcam; dynein cytoplasmic 1 light intermediate chain 1 (DYNC1LI1): anti-DLC-A, cat. no. ab154251, 1:20, Abcam; phospholipase A and acyltransferase 2 (HRASLS2): anti-HRASLS2, cat. no. bs-6013R, 1:1,000, Thermo Fisher Scientific, Inc.] in the blocking solution mentioned above at 4˚C overnight. Endogenous peroxidases in tissue sections were removed by incubation with 0.3% H₂O₂ for 15 min, and these peroxidase-free arrays were further incubated with a biotinylated secondary antibody (either goat anti-rabbit, cat. no. BA-1000-1.5, 1:200, Vector Laboratories, Inc. or rabbit anti-goat immunoglobulin G, cat. no. BA-5000-1.5, 1:200, Vector Laboratories, Inc.) at room temperature for 60 min. The VECTASTAIN ABC system and DAB Substrate kit (both from Vector Laboratories, Inc.) were used to develop the secondary antibodies captured on arrays, according to the manufacturer's instructions. Following hematoxylin (GHS3; 50 ml, Merck KGaA) counterstaining at room temperature for 5 min and slide mounting with Malinol (Muto Pure Chemicals, Co., Ltd.), images were digitalized using a high-resolution scanner (Mirax Scan, Carl Zeiss AG) at the Taiwan Mouse Clinic (Academia Sinica, Taipei City, Taiwan). Two independent pathologists reviewed and evaluated the imaging results. QuPath (Version 0.3.0; https://qupath.github.io) was employed to produce the cell densities of immunoreactive cells per mm² for all target proteins (50 (link)).