Mirax midi system

Paraffin sections were de-waxed and rehydrated. Antigen retrieval was carried out by microwaving the slides in 0.01 M sodium citric acid buffer (pH 6.0) for 30 min. Sections were then immersed for 1 hour in blocking buffer (3% BSA, 0.2% Triton X-100 in PBS), then incubated in primary antibody (in blocking buffer) at 4° C overnight, followed by incubation with secondary antibody at 4° C for 1 hour. Slides were mounted with antifade reagent with or without DAPI (Life Technologies), and then scanned with a high-resolution MIRAX MIDI system (Carl Zeiss) equipped with both bright field and fluorescence illumination. Images were analyzed by the MIRAX Viewer software.
Polyclonal rabbit anti-Scgb1a1 antibody (US Biological, C5828) was used at 1:200 dilution. Goat anti-pro-SPC (Santa Cruz Biotechnology, sc-7706), rabbit anti-Cyp2f2 (Santa Cruz Biotechnology, sc-67283), goat anti-PDPN (R&D Systems, AF3244), monoclonal mouse anti-p63 4A4 (Santa Cruz Biotechnology, sc-8431), rabbit anti-GFP (Abcam, ab290), goat anti-GFP(Abcam, ab5450), and mouse anti-GFP (Abcam, ab1218) were used at 1:50 dilution. Secondary antibodies (including donkey anti-rabbit, anti-goat, or anti-mouse) each with different Alexa Fluor conjugations were all purchased from Life Technologies, and used at 1:200 dilution.

A computer assisted hot spot analysis was performed on whole block sections. Images were acquired with a standard light microscope and a CCD camera or a Mirax Midi system (Zeiss, Jena, Germany) for digital slide scanning and Mirax viewer software (Zeiss). Positive cells were manually selected by a pathologist. Numbers of positive cells per mm² in the area with the densest infiltration were evaluated using the image analysis software “Count” (Biomas, Erlangen, Germany). In CD83 stains positive cells showing the morphology of neoplastic HRS cells were excluded from the analysis. For evaluation of CD123 only cells with plasmacytoid morphology were counted.

MIRAX MIDI system (Carl Zeiss) equipped with bright field and fluorescence illumination was used to scan the stained lung sections. Whole-slide scanned images were captured with Axiocam MR(m) (Carl Zeiss), and converted to TIFF format with Miraxviewer software (3DHISTECH). The images used for quantification were down-sampled eight times from the original images to reduce the computation time and the use of memory space.