Envision flex substrate buffer containing peroxide

For IHC analysis, tissues were fixed and the slides were stained as reported in [47 (link)]. Briefly, tissues were first fixed in formalin and embedded in paraffin (FFPE) and then incubated overnight at 4 °C with anti-SALL4, anti-GLI1, -cleaved Caspase-3 or -Ki67 antibodies. The next day, the slides were incubated for 20 min with secondary antibodies coupled with peroxidase (Dako), which is then detected by the diaminobenzidine (DAB) solution (ScyTek Laboratories, Logan, UT, USA) and the EnVision FLEX Substrate buffer containing peroxide (Dako, Agilent, Santa Clara, CA, USA). Cell quantification was performed on stained sections with NIS-Elements BR 4.00.05 (Nikon Instruments Europe B.V., Florence, Italy) imaging software. Stained slides were scanned using the NanoZoomer S60 Digital slide scanner C13210-01 (Hamamatsu Photo- nics). Scanned images were viewed and captured with Hamamatsu Photonics’s image viewer software (NDP.view2 Viewing software U12388-01) at indicated magnifications.

Formaldehyde-fixed paraffin-embedded (FFPE) tissues and frozen OCT-embedded tissues were cut into 4μm sections for Gli1, Ki67, cleaved Caspase-3, NeuN, and ERAP1 immunohistochemical staining. FFPE slides were deparaffinized and subjected to heat-induced antigen retrieval at low or high pH buffer, whereas frozen tissues were fixed in 4% paraformaldehyde. Slides were blocked for 30 min with 5% PBS/BSA. FFPE slides were incubated with monoclonal antibodies against Gli1, Ki67, cleaved Caspase-3, NeuN, whereas cryostat sections were incubated with monoclonal antibody ERAP1 (4D2, 50 mg/ml overnight 4 °C). This step was followed by incubation for 20 min with secondary antibodies coupled with peroxidase (Dako). Bound peroxidase was detected with diaminobenzidine (DAB) solution and EnVision FLEX Substrate buffer containing peroxide (Dako). Cell quantification was performed on collected sections using the imaging software NIS-Elements BR 4.00.05 (Nikon Instruments Europe B.V., Italy). Images were captured by HistoFAXS software (TissueGnostics GmbH, Vienna, Austria) at 20x magnification. Tumor regions were analyzed with HistoQuest software (TissueGnostics) for automatic color separation and quantification. Expression levels were evaluated as stained area per mm².