Dmi6000 ffw microscope

STn and GLUT1 were evaluated in the same tumor sections by double staining immunofluorescence microscopy. Briefly, FFPE esophageal tumor sections were deparaffinized, rehydrated and incubated for 20 min with 1 mM EDTA pH 9 to recover STn and GLUT1 epitopes. Unspecific background was blocked with Protein Block (Leica Biosystems, Wetzlar, Germany) for 5 min and both markers (STn and GLUT1) were incubated with primary monoclonal antibodies, as previously described for immunohistochemistry staining. Posteriorly, moAb anti-STn and anti-GLUT1 were detected using an Alexa Fluor 488 goat anti-mouse IgG (Thermo Fisher Scientific, Waltham, MA, USA) and an Alexa Fluor 594 goat anti-rabbit IgG (H+L), respectively, at the dilution of 1:100 for 30 min at room temperature. Nuclear counterstaining was obtained using a 4′,6-diamidino-2-phenylindole, dihydrochloride (DAPI; Thermo Fisher Scientific, Waltham, MA, USA) solution for 10 min. Fluorescence images were acquired on a Leica DMI6000 FFW microscope (Leica Microsystems, Wetzlar, Germany) using the Las X software (Leica Microsystems, Wetzlar, Germany).

Glycans and HOMER3 were evaluated in the same tumor sections by double staining immunofluorescence microscopy. Briefly, FFPE bladder tumor sections were deparaffinized and rehydrated, followed by heat-induced epitope retrieval with citrate buffer pH 3.0. Unspecific background was blocked with Protein Block (Leica Biosystems). T antigen was stained using PNA-FITC lectin (Vector Laboratories) and Tn antigen was screened using VVA-FITC lectin (Vector Laboratories) 1:1000 for 1 h at room temperature. Their sialylated counterparts were stained using the same lectins after a 4 h incubation with 0.2 mU/mL α-neuraminidase from Clostridium perfringens (Sigma-Aldrich) at 37 °C. HOMER3 was evaluated using a rabbit polyclonal antibody (PA5–59383, Thermo Fisher Scientific), 1:100 overnight at 4 °C, following detection with an Alexa Fluor 594 goat anti-rabbit IgG (H + L) at the dilution of 1:100 for 30 min at room temperature. Nuclear counterstaining was obtained using a 4′,6-diamidino-2-phenylindole, dihydrochloride (DAPI; Thermo Fisher Scientific) solution for 10 min. Fluorescence images were acquired on a Leica DMI6000 FFW microscope (Leica Microsystems) using the Las X software (Leica Microsystems).