Pannoramic midi 2 slide scanner

To evaluate the impact of TILs, we determined the immunoscores by immunohistochemical staining for CD3 and CD8. We scanned all the immunohistochemically stained slides for CD3 and CD8 using a Pannoramic MIDI II slide scanner (3DHISTECH, Budapest, Hungary). We captured images from two regions—the core of the tumor (CT) and invasive margin (IM)—using the CaseViewer 2.0 software (3DHISTECH). CD3- and CD8-immunoreactive lymphocytes were identified from the captured images using NIH Image Analysis software (version 1.6.0; National Institutes of Health, Bethesda, Maryland, USA) after setting a consistent intensity threshold. The CD3- and CD8-immunoreactive lymphocytes were expressed as pixels in each region. Using the cut-off (median value), the pixel value of each case was classified to high (score 1) and low (score 0). Immunoscores were defined as the sum of the scores of two regions and are divided into high (score 3–4) and low (score 0–2) scores [21 (link)].

In all, 8 µm cryosections were dried and stained with Hematoxylin Phloxine Saffron (HPS) (Dako). For Von Kossa staining, cryosections were incubated 30 min at RT in 1% silver nitrate in the dark followed by 15 min incubation in UV chamber and nuclear fast red counterstain. All sections were analyzed using a Pannoramic Midi II slide scanner and Case Viewer software (3D HISTECH Ltd.).
For immunohistochemical staining, cryosections were fixed in 4% PFA 15 min, incubated in PBS-Triton 0.5% 15 min followed by 1 h in PBS-BSA 1% at RT. Muscle sections were incubated with rabbit polyclonal anti-laminin antibody (dilution 1/200, Abcam) or rabbit polyclonal anti-osteocalcin or anti-Collagen III antibody (dilution 1/100 and 1/200, Abcam) in PBS-BSA 1% overnight at 4 °C. Corresponding normal polyclonal rabbit IgG was used as negative control (Abcam). Sections were washed with PBS and incubated with secondary antibody Donkey anti-rabbit Alexa Fluor 594 or Goat anti-rabbit Alexa Fluor 488 (1/500, Thermo) in PBS-BSA 1% for 1 h à room temperature. Slides were finally mounted in Vectashield antifade mounting medium with DAPI (Vector). All sections were analyzed using inverted epifluorescence microscope (DMi8, Leica) and Fiji software (ImageJ)

J-Lat 1C10 cells were washed with PBS and allowed to adhere to Polylysine coated slides (VWR, Radnor, PA, USA) marked with a hydrophobic barrier using A-PAP pen (Histolab, Gothenburg, Sweden). PLA was performed according to the manufacturer’s protocol (Sigma) with a few modifications: PLA plus and minus probes were diluted 1:20, amplification buffer (5×) was used at 10×. All washes were performed in PBS. Antibodies (1:1,000) were used against Tat (Abcam, Cambridge, United Kingdom), FLAG M2 (Sigma). Before DAPI staining and mounting with Duolink in Situ mounting media with DAPI (Sigma), FITC-conjugated anti-GFP (Abcam) was applied (1:500) for 1 h at ambient temperature protected from light. Slides were sealed with nail polish and stored at 4°C overnight before imaging. Slides were imaged using a Pannoramic Midi II slide scanner (3DHistech, Budapest, Hungary) and images were exported using the CaseViewer application. Images were analyzed with ImageJ (version 2.0.0-rc-69/1.52) and macros developed in house. Each RGB image was separated into separate channels. Based on the blue (DAPI) channel, nuclei were identified. In the red channel, spots (“maxima”) were detected with prominence thresholds 6–48.