Journal: Cancers

Article Title: Spatial Distribution of Non-Immune Cells Expressing Glycoprotein A Repetitions Predominant in Human and Murine Metastatic Lymph Nodes

doi: 10.3390/cancers15235621

Figure Lengend Snippet: Evaluation of GARP in HUVEC, LEC, and HLF cells cultured under basal conditions. ( a ) Western blot analysis of GARP expression. The blot is a representative blot out of 4 independent experiments. The bar graph shows the quantification of GARP protein levels relative to the GAPDH protein signal (GARP/GAPDH signals) ( n = 4, means ± SD, n.s. determined by one-way ANOVA). ( b ) Flow cytometry analysis of GARP at the surface of primary cells. Jurkat cells overexpressing GARP (Jurkat−hGARP) were used as a positive control. The isotype control is represented in grey, and the positive signal is depicted in red as a percentage of the maximum. The relative MFI of GARP in flow cytometry is represented with a bar graph ( n ≥ 3, means ± SD, n.s., no significance, determined by one-way ANOVA).

Article Snippet: Sections were blocked with animal-free blocking solution (15019 L, Cell Signaling), followed by an incubation with either 5 µg/mL of mouse monoclonal anti-human GARP antibody (clone MHG-6 [ ]) in Dako antibody diluent or no primary antibody (negative control) for 90 min. After two washes with PBS supplemented with Tween20 (PBS-T), the EnVision-HRP secondary antibody (K4001, Dako Agilent, Diegem, Belgium) was incubated at room temperature for 30 min. Staining was amplified using tyramide signal amplification working solution (TSA, NEL741001KT, PerkinElmer) containing fluorescein isothiocyanate dye (1:500, FITC) for 10 min, washed thrice with PBS-T.

Techniques: Cell Culture, Western Blot, Expressing, Flow Cytometry, Positive Control

Journal: Cancers

Article Title: Spatial Distribution of Non-Immune Cells Expressing Glycoprotein A Repetitions Predominant in Human and Murine Metastatic Lymph Nodes

doi: 10.3390/cancers15235621

Figure Lengend Snippet: Full scans of Western blot images shown in a. ( a ) Western blot membrane of GARP in Jurkat hGARP used as a positive control. The red rectangle corresponds to the first lane in a. ( b ) Western blot membrane of GARP on HUVECs, LECs, and HLFs in which the red rectangle corresponds to the right part of Western blot shown in a.

Article Snippet: Sections were blocked with animal-free blocking solution (15019 L, Cell Signaling), followed by an incubation with either 5 µg/mL of mouse monoclonal anti-human GARP antibody (clone MHG-6 [ ]) in Dako antibody diluent or no primary antibody (negative control) for 90 min. After two washes with PBS supplemented with Tween20 (PBS-T), the EnVision-HRP secondary antibody (K4001, Dako Agilent, Diegem, Belgium) was incubated at room temperature for 30 min. Staining was amplified using tyramide signal amplification working solution (TSA, NEL741001KT, PerkinElmer) containing fluorescein isothiocyanate dye (1:500, FITC) for 10 min, washed thrice with PBS-T.

Techniques: Western Blot, Membrane, Positive Control