Axio imager z2 upright epifluorescence microscope

Multiplex immunofluorescence (mIF) was carried out on 16 formalin fixed paraffin embedded (FFPE) endemic BL cases (validation cohort 2), belonging to set of samples previously studied and well characterized for EBV latency program [27 ].
Multiplex IF was applied to simultaneously detect the expression of: a) CD68 (Abcam, ab 955, 1:150) and CD163 (Leica Biosystem, 10D6, 1:200); b) PD-L1 (Dako, clone 22C3, 1:100) and CD163 (Leica Biosystem, 10D6, 1:200); c) PD-L1 and EBV-LMP2A (Abcam, clone 15F9, ab59028, 1:200). These double stainings use red and green or magenta and green chromogens. The colour assignment and staining location are: a) CD68 red/membranous; CD163 green/membranous; b) PD-L1, green/membranous and CD163, pink/membranous or PD-L1, green/membranous and CD163, red/membranous; c) PD-L1, red; LMP2A green/ membranous. The staining procedure was established according to previously published work [28 ]. Tissue sections from the same set of cases and without antibody/fluorophore were used as negative control. Multiplex IF staining reaction and image analysis (including quantification of antibodies expression) were performed using the Vectra 2.0 system (PerkinElmer, Waltham, MA) and Tissue FAXSFluo slide scanning system (TissueGnostics, Vienna Austria) based on a Zeiss Axio Imager Z2 upright epifluorescence microscope.

NIH-3T3 or Cos-7 cells were grown on 0.1% gelatin-coated coverslips in 24- or 48-well culture plates for up to 8 days, changing medium (supplemented with ascorbic acid, 50 μg/ml, SIGMA) every 48 hours. The cells were gently removed as described^{12 (link)} or alternatively by incubating cells with 20 mM NH₄OH, 5 mM EDTA at RT with gentle swirling of the plate so that the intact ECM remained on the coverslips. ECM was incubated with 4 μg/ml of pro-VEGF-C or 4-fold molar excess of other VEGF-C proteins overnight at 4 °C followed by fixation with 4% PFA and blocking in 1% BSA in PBS for 1 hour at RT. The cover slips were then stained by immunohistochemistry using anti-VEGF-C antiserum 6 (1:200) and anti-fibronectin (1:200) followed by Alexa 488 conjugated secondary antibody. Fluorescent images were obtained with an AxioImager.Z2 upright epifluorescence microscope (Carl Zeiss AG, Oberkochen, Germany).