Tumor tissues in the PDX assays and MiniPDX assays were fixed in buffered 10% formalin and routinely stained with hematoxylin and eosin (H&E) and examined by a certified pathologist.
For immunofluorescence studies, cellularized tumor cells (2 × 104 cells, 200 L) were cytospun onto a slide, fixed with 4% paraformaldehyde for 20 min, permeabilized with 0.3% Triton X-100 in PBS for 30 min, and then blocked with 5% normal goat serum for 1 h at room temperature. The cells were then divided into three fractions and incubated with primary mouse monoclonal antibodies at 4 °C overnight against the following proteins: pan-cytokeratin, indicating carcinoma components [27 (link), 28 (link)] (1:200, AE1/AE3, sc-81714, Santa Cruz Biotechnology, Santa Cruz, CA, US), E-cadherin, generally found in gastric adenocarcinomas [29 (link)] (1:50, HECD-1, ab1416, Abcam, Cambridge, UK), and MG7, a marker of gastric cancer [30 (link)] (1:300, NOTA-MG7) [30 (link)]. Subsequently, the cells were probed with secondary antibody donkey anti-mouse IgG H&L (Alexa Fluor® 488) (1:200, ab150105, Abcam). Finally, the cells were mounted with DAPI-containing mounting medium (S36973, Thermo Fisher, MA, US). Images were captured with a fluorescence microscope (Leica, Germany) with Leica Application Suite V4 software and edited with Photoshop (Adobe, US).
For immunofluorescence studies, cellularized tumor cells (2 × 104 cells, 200 L) were cytospun onto a slide, fixed with 4% paraformaldehyde for 20 min, permeabilized with 0.3% Triton X-100 in PBS for 30 min, and then blocked with 5% normal goat serum for 1 h at room temperature. The cells were then divided into three fractions and incubated with primary mouse monoclonal antibodies at 4 °C overnight against the following proteins: pan-cytokeratin, indicating carcinoma components [27 (link), 28 (link)] (1:200, AE1/AE3, sc-81714, Santa Cruz Biotechnology, Santa Cruz, CA, US), E-cadherin, generally found in gastric adenocarcinomas [29 (link)] (1:50, HECD-1, ab1416, Abcam, Cambridge, UK), and MG7, a marker of gastric cancer [30 (link)] (1:300, NOTA-MG7) [30 (link)]. Subsequently, the cells were probed with secondary antibody donkey anti-mouse IgG H&L (Alexa Fluor® 488) (1:200, ab150105, Abcam). Finally, the cells were mounted with DAPI-containing mounting medium (S36973, Thermo Fisher, MA, US). Images were captured with a fluorescence microscope (Leica, Germany) with Leica Application Suite V4 software and edited with Photoshop (Adobe, US).
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