Ultravision lp detection system ap polymer fast red chromogen assay

Paraffin-embedded intestinal equivalents were rehydrated. Sections (4 μm thick) were stained with hematoxylin and eosin (H&E) and thiazine staining (Bio-Optica^®, Milan, Italy). Occludin tissue expression was detected using a mouse anti-occludin primary antibody (Novus). Immunohistochemistry was performed using Fast Red chromogen. Stainings were performed according to the UltraVision LP Detection System AP Polymer & Fast Red Chromogen assay (Thermo Fisher Scientific, Waltham, MA, USA). Negative controls were obtained by omitting the primary antibody. The expression intensity was quantitatively determined using ImageJ software (Wayne Rasband). Signal detection was performed on at least 3 different sections and the results were reported as a mean.

Cryosections (4 µm) of skin from healthy donor and from pemphigus patients were methanol-fixed and rehydrated in PBS. The staining was performed using the UltraVision LP Detection System AP Polymer & Fast Red Chromogen assay (Thermo Fisher Scientific), according to the manufacturer’s instructions. Briefly, slides were treated with Ultra V Block, and samples were incubated with mouse anti-CD95/Fas Ab (UB2; Immunotech, Marseille, France) for 1 h at room temperature. After washes in PBS, Primary Antibody Enhancer (Thermo Fisher Scientific) was added for 20 min at room temperature, followed by incubation with AP Polymer anti-mouse/rabbit IgG for 30 min at room temperature. Slides were stained with Fast Red using Naphthol Phosphate as substrate. Samples were analyzed under a conventional optical microscope (Zeiss Axioskope 40).

cSCC skin reconstruct were fixed with formalin for 2 h at room temperature, dehydrated and embedded in paraffin. The staining was performed using the UltraVision LP Detection System AP Polymer & Fast Red Chromogen assay (Thermo Fisher Scientific, Waltham, MA, USA), according to the manufacturer’s instructions. Briefly, slides were treated with Ultra V Block and samples were incubated with anti-Ki-67 (1:200; Epitomics, Burlingame, CA, USA), or anti-keratin (ready to use; Cell Marque), or anti-MMP9 (1:100; Abcam, Cambridge, UK) or anti-psoriasin (1:50, Abcam, Cambridge, UK) for 1 h at room temperature.
After washes in PBS, Primary Antibody Enhancer (Thermo Fisher Scientific, Waltham, MA, USA) was added for 20 min at room temperature, followed by incubation with AP Polymer anti-mouse/rabbit IgG for 30 min at room temperature. Slides were stained with Fast Red using Naphthol Phosphate as substrate. Samples were analyzed under a conventional optical microscope (Zeiss Axioskope 40, Carl Zeiss, Jena, Germany).