Envision horseradish peroxidase kit

Immunohistochemical methods were used to evaluate expression of JAK3, STAT2, STAT4, and STAT6 in both lesional and perilesional skin and compared with healthy control skin. Paraffin-embedded tissue sections were mounted onto SuperFrost slides, deparaffinised, then treated in a solution of TRS, and transferred to distilled water. Endogenous peroxidase activity was blocked by 0,3% hydrogen peroxide in distilled water, and then sections were rinsed with Tris-buffered saline (TBS, Dako, Denmark) and incubated with primary rabbit polyclonal antibody against STAT2 (Santa Cruz Biotechnology Inc.), mouse monoclonal antibody against STAT4 (Santa Cruz Biotechnology Inc.), and primary rabbit polyclonal antibody against STAT6 (Santa Cruz Biotechnology Inc.) and incubated overnight with mouse monoclonal antibody against JAK3. Immunoreactive proteins were visualized using EnVision-horseradish peroxidase kit (Dako, Carpinteria, CA, USA) according to the instructions of the manufacturer. Visualisation was performed by incubating the sections in a solution of 3,3′-diaminobenzidine (DakoCytomation, Denmark). After washing, the sections were counterstained with hematoxylin and coverslipped. For each antibody and for each sample, a negative control was processed.

Paraffin-embedded tissue sections were mounted onto Superfrost slides, deparaffinized, then treated in a solution of TRS (Target Retrieval Solution, Dako, Glostrup, Denmark) and transferred to distilled water. Endogenous peroxidase activity was blocked and then sections were rinsed with Tris-buffered saline (TBS, Dako, Glostrup, Denmark), and incubated with primary rabbit polyclonal antibody against STAT2 mouse monoclonal antibody against STAT4 (primary rabbit polyclonal antibody against STAT6 and incubated overnight with mouse monoclonal antibody against JAK3 (Santa Cruz biotechnology Inc, Dallas, TX, USA). Immunoreactive proteins were visualized using EnVision-horseradish peroxidase kit (Dako, Carpinteria, CA, USA). After washing, the sections were counter-stained with hematoxylin and cover slipped. For each antibody and for each sample a negative control were processed. The expression of STAT2, STAT4, and STAT6 are expected to be cytoplasmatic and nuclear upon activation.

Formalin‐fixed paraffin‐embedded tissue was stained as described previously (Mizee et al., 2014). In short, deparaffinization and antigen retrieval, sections were incubated overnight with appropriate antibodies (for details, see Table 2) and subsequently incubated with horseradish peroxidase EnVision kit (Dako, Denmark) followed by 3,3’diaminobenzidine‐tetrahydrochloride dihydrate (Dako, Denmark). All sections were counterstained with hematoxylin after which they were analyzed with a light microscope (AXIO Scope A1, Carl Zeiss, Germany). For cellular localization studies, sections were incubated overnight with appropriate antibodies followed by incubation with Alexa‐488‐labeled goat antimouse IgG1 (1:200; Molecular Probes, USA) and analyzed by confocal microscopy (Leica DMI 6000 SP8, Leica, Germany).