Diaminobenzidine dab solution

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.

Tissues used for immunohistochemical staining were fixed in formalin and paraffin-embedded (FFPE). FFPE slides were incubated with monoclonal antibodies against RIG-I or MEX3A (1:100, overnight, at 4 °C). The day after, the slides were incubated for 20 min, with secondary antibodies coupled with peroxidase (Dako). Bound peroxidase was detected by diaminobenzidine (DAB) solution (ScyTek Laboratories, Logan, UT, USA) 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., Florence, Italy). Images were captured by HistoFAXS software (TissueGnostics GmbH, Vienna, Austria), at 20× magnification.