Benchmark ultra autostainer

Our experiments were performed on two samples of human granulation tissue. The first sample was a set of eight consecutively sliced tissue sections of H&E-stained granulation tissue. The second one was a set of ten consecutive tissue sections of a CD31-stained granulation tissue. The thickness of the sections was 4 µm for the H&E and 3 µm for the CD31 samples. For both staining methods, the fresh tissue was first fixed in 5% formalin and then embedded in paraffin. The formalin-fixed paraffin-embedded (FFPE) tissue blocks were cut into consecutive sections and mounted onto glass slides. For the H&E staining, the tissue was deparaffinized, rehydrated and then stained in Mayer’s hematoxylin and eosin. The immunohistochemical staining of CD31 was performed with the help of a BenchMark Ultra autostainer (Ventana Medical Systems, Oro Valley, AZ, USA) employing a mouse anti-CD31 antibody (1:40; clone JC70A; Dako, Glostrup, Denmark). After staining both the H&E and CD31, the slides were covered with glass cover slips and were scanned as whole slide images (WSI) via the PreciPoint M8 slide scanner (PreciPoint GmbH, Freising, Germany) with a 20× objective (Olympus UPlan FLN 20×, Olympus, Tokyo, Japan). The resolution observed at maximum zoom was 0.28 µm per pixel.

IHC staining for HLA-DR was performed as previously described^{3 (link)}. Briefly, 4 µm FFPE sections were stained using an HLA-DR antibody (Santa Cruz Biotechnology (sc-53319) in a Ventana BenchMark™ ULTRA autostainer. Sections were de-paraffinized and subject to antigen retrieval using Cell Conditioning 1 (CC1, pH 8.5) for 64 minutes at 95 °C. The primary antibody was applied at a concentration of 1:1000. Interpretation was performed as previously described^{3 (link)}.

Sections of 3 µm-thick, formalin-fixed, paraffin-embedded tissue were cut and placed on Superfrost Plus microscope slides. The slides were heated for one hour at 60 °C in a dry oven to enhance tissue attachment and soften the paraffin. The immunohistochemistry procedure was performed on a Ventana BenchMark ULTRA autostainer using the standard protocol. In summary, the sections underwent deparaffinization, rehydration, and antigen retrieval using CC1 (prediluted, PH 8.0) antigen retrieval solution (Ventana). The primary antibodies were added and incubated with the sections at a dilution suggested by the manufacturer. The following primary antibodies were used: mouse monoclonal anti-human SerpinB3/SCCA [clone OTI1A5] (ab180396, Abcam, Cambridge, MA, USA, 1:100 dilution) and rabbit monoclonal anti-human α 1 Antitrypsin (Serpina1) [clone EPR17087-50] (ab207303, Abcam, Cambridge, MA, USA, 1:400 dilution). The visualization process was performed using the Ultraview universal DAB IHC detection kit, and it was afterwards counterstained using hematoxylin and bluing solution. The slides were gently cleaned, dehydrated in graded ethanol and xylene, and then mounted using mounting media on a microscope slide.