MELC image acquisition

We generated the multiplexed histology data on a modified Toponome Image Cycler^® MM3 (TIC) originally produced by MelTec GmbH & Co.KG Magdeburg, Germany^57,58. The robotic microscopic system consists of: (i) an inverted widefield (epi)fluorescence microscope Leica DM IRE2 equipped with a CMOS camera and a motor-controlled XY-stage, (ii) CAVRO XL3000 Pipette/Diluter (Tecan GmbH, Crailsheim, Germany), and (iii) a software MelTec TIC-Control for controlling microscope and pipetting system and for synchronized image acquisition. The MELC run is a sequence of cycles, each containing the following four steps: (i) incubation of the fluorescence-coupled antibody and subsequent washing; (ii) cross-correlation-based auto-focusing; (iii) photo-bleaching of the fluorophore; and (iv) a second autofocusing step followed by acquisition of a 3D stack post-bleaching fluorescence image. In each four-step cycle another fluorescence-labeled antibody is used. After the sample was labeled sequentially by DAPI (Roche, Cat. No. 10236276001, dilution 1:5000) and all antibodies of interest as described above, the experiment was completed. Fluorescence-coupled antibodies used: IgA2-PE (Clone REA995, Miltenyi, Cat. No. 130-117-763, dilution 1:50); CD27-PE (Clone REA499, Miltenyi, Cat. No. 130-114-166, dilution 1:50); CD38-PE (Clone IB6, Miltenyi, Cat. No. 130-113-427, dilution 1:50); and IgA-PE (Clone IS11-8E10, Miltenyi, Cat. No. 130-114-002, dilution 1:50). The antibodies were stained in the indicated order.