Immunohistochemical Analysis of HBS1 Keratin Expression

Immunohistochemistry was performed according to published protocols (Mlitz et al. 2014 ; Ehrlich et al. 2019 (link)) with modifications. In brief, antigens were demasked with citrate buffer (pH 6), and the samples were incubated with an antiserum against HBS1 keratin (dilution 1:500). Biotinylated sheep anti-mouse immunoglobulin (RPN1001V, lot 9793564, GE Healthcare, Chalfont, UK) at a dilution of 1:100 was used as secondary antibody, and sheep serum (10%) was added to prevent unspecific binding. In control experiments, the primary antibody was replaced by serum of the same mouse prior to immunization (preimmune serum) at the same dilution. 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.