Anti dig clone 9h27l19

The labeling of antibodies with digoxigenin (DIG) was performed similarly as IR700 conjugation using 1 mg of anti-CD29, anti-CD44 or isotype rat IgG2b (clone KM16, IM7, and LTF-2, respectively, all purchased from Bio X Cell) and 50 μg of digoxigenin succinimidyl ester (Thermo Fisher Scientific). The resulting DIG-labeled antibodies were abbreviated as anti-CD29-DIG, anti-CD44-DIG, and isotype-DIG, respectively. Each DIG-labeled antibody (0.335 nmol in 200 μL PBS) was injected intravenously into tumor-bearing mice, tumors and contralateral skin ware harvested a day later. The distribution of labeled antibodies was detected in FFPE sections by anti-DIG IHC using anti-DIG (clone 9H27L19; ThermoFisher Scientific).

Multiplex immunohistochemistry was carried out as described previously
¹⁸ (link) using an Opal Automation IHC Kit (Akoya Bioscience) and Bond RXm autostainer (Leica Biosystems). The sections were stained with DAPI and the following Abs: anti‐CD45 (clone D3F8Q; Cell Signaling Technology; 1:500), anti‐DIG (clone 9H27L19; Thermo Fisher Scientific; 1:500), anti‐Ki‐67 (clone D3B5; Cell Signaling Technology; 1:500), and anti‐pCK (rabbit poly; Bioss Antibodies; 1:250). Stained slides were mounted with ProLong Diamond (Thermo Fisher Scientific) and imaged with a Mantra Quantitative Pathology Workstation (Akoya Biosystems). The obtained images were analyzed with inForm Tissue Finder software (Akoya Biosystems). To calculate the percentage of Ki‐67 positive cancer cells, inForm software was trained to detect tissue and cell phenotypes using machine‐learning algorithms based on the following criteria: areas with pCK expression = tumor, other areas = stroma, pCK+ CD45− cells = cancer cells, pCK− CD45+ = blood cells, and pCK− CD45− = other cells. inForm software computed the percentage of Ki‐67‐positive cells among cancer cells. The average percentage was calculated from five images for each specimen.