Tissuefaxs plus system

Twenty-Six human embryos and foetuses (GW09 x2, GW10 x3, GW11x2, GW12x3, GW13 x3, GW14 x2, GW15 x2, GW16 x3, GW17 x3, GW18 x3 and GW19 x1 as biological/technical replicates were done via sectioning) were used for this study. Tissue preparation, immunohistochemistry and fluorescence-based immunohistochemistry procedure on human cochlear section was described in previous publications (29 (link)–32 (link)).
Positive controls (e.g., small intestine, brain, and pancreas) were supplemented to each experiment. Negative controls are acquired by alternating the primary antibodies with reaction buffer or substituting them with isotype-matching immunoglobulins. Immunohistochemistry sections were digitally examined using a Zeiss AxioVision 4.1 microscope software coupled to an AxioCam HRc colour camera and an AxioPlan2 microscope (Zeiss, Jena, Germany). The fluorescence immunohistochemistry stained sections were digitalized at 40x and 63x magnifications using a TissueFAXS Plus System coupled onto a Zeiss^® Axio Imager Z2 microscope (Jena, Germany). Image acquisition was performed using the TissueFAXS software (TissueGnostics^®, Vienna, Austria). The quantitative analysis of the amount and distribution of macrophages/monocytes was performed in GraphPad prism 9.

The immunostained and riboprobes reacted sections were digitalized at 20× magnification utilizing a TissueFaxs Plus System coupled onto a Zeiss^® Axio Imager Z2 Microscope (Jena, Germany). Regions of interest were then acquired using the TissueFaxs (TissueGnostics^®, Vienna, Austria). TrkB immunostaining was scored [50 (link)] (0: no staining, low (1): under 30% of cells positive, middle (2): 30–66% of cells positive, high (3): more than 66% of cells positive in cancer cell nests) and Mann–Whitney test was used to detect differences between HNSCC and UPPP. Dot Plots, frequency diagrams, and heat maps were created using TissueQuest (TissueGnostics^®) [51 (link),52 (link)].

The immunostained and riboprobes reacted sections were digitalized at 20× magnification utilizing a TissueFaxs Plus System coupled onto a Zeiss^® Axio Imager Z2 Microscope (Jena, Germany). Regions of interest were then acquired using the TissueFaxs (TissueGnostics^®, Vienna, Austria). The intensity of signals localized onto the affixed sections was then evaluated using HistoQuest^® (TissueGnostics) software. Details of the intensity quantification are given in Supplementary Information, Supplementary Methods.
Alternatively, p75NTR and NTRK1 immunostaning were scored (0): no staining; low (1): under 30% of cells positive; middle (2): 30–66% of cells positive; and high (3): more than 66% of cells positive in cancer cell nests) and Mann–Whitney test was used to detect differences between HNSCC and UPPP. p75NTR staining was also evaluated as staining present (1) or absent (0).