3 amino 9 ethylcarbazole

For paraffin sections, pancreata were processed as previously described (Lattke et al. 2012 (link), Salem et al. 2014 (link)). Sections were incubated with primary antibodies overnight (Supplementary Table 2). Secondary antibodies were coupled with Alexa Fluor (Invitrogen) for immunofluorescence or with horseradish peroxidase that was developed by 3-amino-9-ethylcarbazole (DakoCytomation) for immune-histochemistry. Fibrosis was assessed using Pikro-Siriusrot solution (Morphisto, Frankfurt, Germany). Immunofluorescent stainings were visualized as before (Lattke et al. 2012 (link)), and other stainings were analysed on a Leica DM IRB microscope (Leica Microsystems) equipped with ProgRes C14 digital camera (Jenoptik).

Immunohistochemical stainings were performed according to published protocols (Mlitz et al. 2014 (link); Alibardi et al. 2016 (link)). In brief, tissues were sectioned at 4-μm thickness and antigens were demasked with citrate buffer, pH 6 (Dako). Endogenous peroxidase was blocked with hydrogen peroxide. Mouse anti-EDDM antiserum at a dilution of 1:200 was used as primary antibody. Biotinylated sheep anti-mouse immunoglobulin (RPN1001V, lot 9793564, GE Healthcare, Chalfont, UK) at a dilution of 1:200 was used as secondary antibody, and sheep serum (10%) was added to prevent unspecific binding. In control experiments, the primary antibody was replaced with preimmune serum. The samples were incubated with streptavidin-biotin-horseradish peroxidase (HRP) complex and 3-amino-9-ethylcarbazole (DakoCytomation, Glostrup, Denmark) to develop red color. Nuclei were counterstained with hematoxylin.

5 µm sections of paraffin embedded skin samples were air-dried overnight at 37 °C, dewaxed and rehydrated. Stainings were performed by an automated BOND system (Leica) according to the manufacturer’s instructions: epitope retrieval was performed at pH6 in epitope retrieval solution (DAKO) and incubated with goat anti-human IL-17A (#AF-317-NA, R&D Systems) followed by a biotinylated anti-goat secondary antibody (#BA-9500-1.5, Vector Laboratories). For detection of specific binding, streptavidin peroxidase and its substrate 3-amino-9-ethyl-carbazole (DAKO) were used. All slides were counter stained with hematoxylin. Stainings without primary antibodies were used as negative control. Positive cells were counted in four to nine visual fields per condition.

Immunohistochemical analyses were performed on 3 μm thick sections in all tumours classified as microsatellite-unstable by polymerase chain reaction-based methods. Briefly, the slides were pretreated by boiling for 10 min with a microwave in target retrieval buffer (pH 9, Dako, Hamburg, Germany) before application of monoclonal antibodies specific for MSH2 (clone FE11, dilution 1:100, Calbiochem, Darmstadt, Germany), MLH1 (clone G168-15, dilution 1:100, BD Biosciences, San Diego, CA, USA), MSH6 (1:200; clone 44; BD Biosciences, San Diego, CA, USA), and PMS2 (ready to use; clone EPR3947, Cell Marque, Sigma Aldrich, St. Louis, Missouri, USA). An immunoperoxidase method was used to visualise bound antibodies with 3-amino-9-ethylcarbazole (Dako) as chromogen.

Six fresh normal skin tissues, including paired exposed and unexposed areas, were available. According to the streptavidin peroxidase-conjugated method, the skin tissue samples were fixed in 10% formalin solution for 6–12 h at room temperature and embedded in paraffin. Formalin-fixed sections, with 4 μm thickness, were stained with hematoxylin and eosin for further study. The sections were incubated with rabbit anti-human RGR monoclonal antibody (cat. no. PA5-100993; 1:200; Invitrogen Biosciences) at 4°C overnight, incubated with horseradish peroxidase-conjugated IgG goat anti-rabbit secondary antibody (1:150; DAKO, Glostrup, Denmark) for 30 min, and finally visualized with 3-amino-9-ethylcarbazole (DAKO). The immunohistochemical scores were determined in a blinded manner under a light microscope. The staining intensity and percentage of stained keratinocytes in five fields of sections were qualitatively scored using the scores of 3+ (strong), 2+ (moderate), 1+ (weak), and 0 (negative) staining for each case, as described previously. The overall RGR expression score was obtained by summing the staining intensities and corresponding percentages according to the calculation formula (3 × x% + 2 × x% + 1 ×x% = total score) (Toyama et al., 2019 (link)).

Necropsies were performed according to standard procedures under BSL-3 conditions. Specimens of brain, heart, lungs, liver, spleen, kidneys and variably also intestine were fixed in 4% neutral buffered formaldehyde, processed, embedded in paraffin wax, sectioned at 2–4 µm thickness and stained with hematoxylin and eosin.
Immunohistology was performed on the following sections for the ZH501-infected agama with the avidin–biotin–peroxidase complex method (Elite PK6100 kit; Vector Laboratories, Burlingame, CA, USA), citric buffer antigen retrieval, 3-amino-9-ethylcarbazole (Dako, Glostrup, Denmark) as chromogen and hematoxylin counterstain. A mouse hybridoma supernatant containing IgG1 directed against the Gc protein of RVFV (clone9A9) was used as primary antibody in a dilution of 1:1000. MP-12-infected and -uninfected Vero 76 cell pellets served as positive and negative controls, respectively. Further negative controls consisted of replacement of the primary antibody by tris buffered saline on serial sections.