Mayer s hematoxylin staining

After deparaffinization and antigen unmasking with citrate buffer (pH6.0 for 30 min in 98 °C) the sections were fixated in cold acetone for 5 minutes (−20 °C). Then the endogenous peroxide activity was blocked by the incubation in 3% methanolic hydrogen peroxide (30 minutes) followed by blocking of unspecific binding by Normal Horse Serum (Vector Laboratories, Burlingame, CA, USA). After washing in phosphate-buffered saline anti-OSTβ (sc-163192, Santa Cruz) and anti-ASBT (sc-27493, Santa Cruz) were used as primary antibodies and biotynaleted anti-mouse/anti-rabbit IgG (BA-1400, Vector Laboratories) served as secondary antibody. Reactions were visualized using ABC Vectastain and DAB kits (Dako, Agilent Technologies, Denmark). Additionally, tissue structures were visualized by Mayer’s Hematoxylin staining (DAKO). The negative controls, in which the primary antibodies were omitted, were included in the study and uniformly demonstrated no reaction. Images were acquired with ZEISS Axio Imager Z2 microscope equipped with Zen Pro 2011acquisition program.

Frozen liver sections were fixed by methanol and acetone mixture (1:1) at −20 °C for 5 minutes. Immunofluorescence analyses of the examined proteins were carried out with rabbit anti-FGFR4 (8562, Cell Signaling) and mouse anti-FGF19 (MAB969, R&D Systems) antibodies. Then incubation with either Fluorescein(FITC)-conjugated anti-rabbit IgG (711-095-152, Jackson ImmunoResearch) or Rhodamine Red^TM-X-conjugated anti-mouse IgG (715-295-150, Jackson ImmunoResearch) was performed. Vectrashield Mounting Medium with DAPI (H-1200, VECTOR) were used to envision cell nuclei. The negative controls, in which the primary antibodies were omitted, were included in the study (data not shown). Additionally, liver tissue structures were visualized by Mayer’s Hematoxylin staining (DAKO). Images were acquired with ZEISS Axio Imager Z2 fluorescence microscope equipped with Zen Pro 2011 acquisition program.

The localization and level of PDC-E2 and p-STAT3 were examined in frozen liver sections (6 µm) using immunohistochemistry. Briefly, frozen sections were fixed with acetone at -20 °C for 5 min. Then, they were incubated with 3% H₂O₂ followed by exposure to the Avidin/Biotin blocking kit (Vector Laboratories, #SP-2001). After blocking with 10% NHS (Normal Horse Serum) for 30 min, the sections were probed with primary antibodies: anti-PDC-E2 (Santa Cruz, #271534, diluted 1:250) and p-STAT3 (Y705; Cell Signaling, #4113, diluted 1:100) for 24 h at 4 °C. Next, biotinylated anti-mouse/anti-rabbit IgGs (#BA-1400, Vector Laboratories) were used as secondary antibodies. The reactions were visualized using the ABC Vectastain and DAB kit (Dako, Agilent Technologies, Denmark). Additional counterstaining with Mayer’s Hematoxylin staining (DAKO) was also performed. The negative control, in which the primary antibodies were omitted, was included in the study and uniformly demonstrated no reaction. To confirm the membrane localization of PDC-E2, an immunofluorescent assay was performed as described previously^{24 (link)}. Images were acquired with a ZEISS Axio Imager Z2 fluorescence microscope.