Mayer s hematoxylin

Cryostat sections (6–7 μm) of gastrocnemius or soleus muscle were fixed with 4% paraformaldehyde/phosphate buffered solution (PFA/PBS; 10 min, RT). Endogenous peroxidase was blocked with 3% H₂O₂. Non-specific sites were blocked with 1% normal porcine serum (Dako Deutschland GmbH, Hamburg, Germany) in PBS. Table A (in S1 File), shows the primary monoclonal and polyclonal antibodies used in this study.
Single staining was performed by incubation of the sections with a primary antibody and thereafter with goat anti-rabbit horseradish peroxidase (HRP)-conjugated (LINARIS GmbH, Dossenheim, Germany) or polyclonal goat anti-rat HRP-conjugated (AbD Serotec, Kidlington, UK) antibodies; endogenous peroxidase activity was suppressed with 3% H₂O₂ in PBS; afterwards the sections were incubated with with 3’-diaminobenzidine (DAB) solution (Roche Diagnostics). Nuclei were counterstained with Mayer's Hematoxylin (Carl Roth GmbH & Co. KG, Karlsruhe, Germany) or Hoechst 33342 (Invitrogen, Eugene, USA). Finally, sections were photographed using an Axioplan2 imaging microscope (Carl Zeiss GmbH) and the digital high resolution imaging system AxioCam/AxioVision (Carl Zeiss GmbH). The quantification of the immunohistochemistry (IHC) was performed using the AxioVision Release 4.8.2 software (Carl Zeiss GmbH) or the software ImageJ (Scion ImageJ, National Institutes of Health, Bethesda, USA).

For IL1β and CD68 immunohistochemistry, cryostat sections (7 μm) were fixed with 4% PFA/PBS(10 min, ambient temperature). Non-specific sites were blocked with 1% normal swine serum (Dako Deutschland GmbH, Hamburg, Germany) in PBS. Single staining was performed by incubation of polyclonal anti- IL1ß (1:100: Abcam; Cambridge, UK) or monoclonal anti-CD68 primary antibody (1:50; AbD Serotec, Kidlington, UK) with goat polyclonal HRP-conjugated anti-rabbit (1:200; LINARIS GmbH, Dossenheim, Germany) or anti-rat (1:100; AbD Serotec, Kidlington, UK) antibody; endogenous peroxidase activity was suppressed with 3 % H₂O₂ in PBS; afterwards, the sections were incubated with DAB solution (Roche Diagnostics). Nuclei were counterstained with Mayer’s hematoxylin (Carl Roth GmbH & Co. KG, Karlsruhe, Germany).

Tissue slides were deparaffinized with xylene twice for 10 min each, and rehydrated with alcohol in four baths of decreasing concentrations (100%, 96%, 70% and 50%) for 3 min. each, followed by washing with distilled water and rinsed in TBS three times for 3 min. Antigen retrieval was done using heat induced epitopes retrieval (HIER) microwave oven method. Therefore the slides were cooked in 10 × concentrated citrate buffer (pH 6.0) (DCS Innovative Diagnostik-Systeme, Hamburg, Germany) for 30 min. After cooling down, the slides were covered with 3% hydrogen peroxide for 10 min. to block endogenous peroxidases activity followed by incubation with the specific primary antibodies anti-phospho-GSK3α/β (Y279/Y216) (#ab68476) (Abcam, Cambridge, UK) (1:100) and anti-phospho-GSK3α/β (Ser21/9) (#9331) (Cell Signaling, Danvers, MA, USA) (1:50) over night at 4° C. For negative control, antibody dilution buffer (DCS Innovative Diagnostik-Systeme) was used instead of the primary antibody. The conventional labeled-streptavidin-biotin method with horseradish-peroxidase and 3-amino ethylcarbazole as chromogen was used for detection according to the manufacturer’s instructions. Afterwards the slides were counterstained with Mayer’s Hematoxylin (Carl Roth, Karlsruhe, Germany) and mounted in Faramount Aquaeous Mounting Medium (Dako, Hamburg, Germany).

For immunohistochemistry (IHC), the avidin–biotin–peroxidase complex (ABC) method using a mouse monoclonal TBEV E protein-specific antibody (clone 19/1493, 1:2,000, kindly provided by Matthias Niedrig) was performed as described previously (44 (link), 45 (link)). 3,3′-diaminobenzidine tetrahydrochloride (DAB) served as a chromogen, and nuclei were counterstained with Mayer’s hematoxylin (Carl Roth GmbH + Co. KG, Karlsruhe, Germany). The olfactory bulb, cerebral cortex, and hippocampus were examined with respect to TBEV-positive cells [single immunopositive cells: 1-5 cells per high power field (HPF); low numbers of immunopositive cells: 6-10 cells per HPF; moderate numbers of immunopositive cells: 11-15 cells per HPF; high numbers of immunopositive cells: >15 cells per HPF]. The distribution of TBEV immunoreaction within the brain regions was evaluated as either focal, multifocal, or diffuse. In the intestine, TBEV-positive cells located in the submucosal and myenteric plexus were evaluated analogously.