3 3 diaminobenzidine (dab)

Immunohistochemistry was performed as previously reported [17 (link)] using an anti-hERG1 monoclonal antibody directed against the S5-pore region (Dival Toscana Srl, Sesto Fiorentino, Italy) at 1:200 dilution. Immunohistochemistry for hERG1B was performed following the same protocol with the exception of permeabilization, carried out by adding Triton X-100 to the blocking solution. The antibody (purified polyclonal anti-hERG1B antibody, Dival Toscana Srl, Sesto Fiorentino, Italy) was used at a final dilution 1:1000. Slides were incubated overnight at 4°C and immunostaining was performed with a commercially available kit (PicTure Max kit and DAB, Invitrogen; Carlsbad CA, USA).

Paraffin-embedded tissues were incubated with AFP (1:100, Abcam, Cambridge, UK), Ki67 (1:200, Abcam, Cambridge, UK) and PCNA (1:200, Abcam, Cambridge, UK). Slides were dried, dewaxed and rehydrated. DAB (Invitrogen, Carlsbad, CA, USA) substrate chromogen solution was applied, followed by counterstaining with haematoxylin.

Ovarian tumor tissues from mice were formalin-fixed and paraffin-embedded. After rehydration and antigen retrieval, the slides (thickness = 4 μm) were incubated with primary antibodies: anti-Ki-67, anti-Bip, anti-phos-S6, anti-phos-AKT and anti-VEGF. The staining was visualized using DAB (Invitrogen, CA). The slides were scanned by Motic (Warrendale, PA). The expression analysis of Ki67, Bip, phos-S6, phos-AKT and VEGF were extracted using the following Image-Pro software (Rockville, MD). Images of the areas of expression were selected for clarity from at least five fields for each IHC specimen. The positive cells were detected, quantified and averaged automatically in selected areas. Each group was measured in seven IHC slides.

For immunohistochemical examinations, 5 µm thick sections were taken from paraffin-embedded brain tissue samples and incubated overnight in a 56°C oven. Subsequently, deparaffinization with toluene (Merck Millipore, Darmstadt, Germany) and rehydration with graded series of ethanol (70-100%) were boiled in citrate buffer (pH 6, Invitrogen) for antigen recovery. The sections were then exposed to H₂O (Abcam, Cambridge, USA) to remove endogenous peroxidase activity. In order to prevent nonspecific binding, the blocking antibody (Invitrogen) was used for 10 minutes. The incubated sections were incubated overnight at + 4°C in EAAT2 / GLT1 antibody (Novus Biologicals) prepared with antibody dilution solution (Invitrogen) at room temperature. Biotinylated secondary antibody (Invitrogen) against the species from which the primary antibody is produced is administered for 10 minutes. and finally treated with HRP-streptavidin (Invitrogen) for 10 minutes. Staining reactions were visualized using DAB (Invitrogen), then counterstained with hematoxylin. Sections were examined using a ×200 objective on a BX-51 Olympus microscope (Olympus, Tokyo, Japan) and photographed. EAAT2/GLT1 immunoreactivity were semi-quantitatively evaluated using a histological score (HSCORE) value, average score was used for statistical analysis, described previously.^{17 (link)}

Multiscreen plates (Millipore) were coated with anti-human IFN-γ antibody (2 µg/ml) or anti-human IL-4 antibody (5 µg/mL; all from BD). After washing and blocking with culture medium, duplications of 5×10⁵ fresh PBMCs were added with concanavalin A (5 µg/mL; Sigma), inactivated EV71/E59 (50 µg/ml) or 5 µg/mL of VP1, VP2 or VP3 peptide mixture prepared from a series of 15-mer peptides with 10-mer overlapping to cover whole VP1 (1–295 aa), VP2 (1–254 aa) or VP3 (1–242 aa), respectively [20] (link), [24] (link). After 40 hours of incubation, the plates were washed and incubated with a biotinylated antibody against IFN-γ (1 µg/mL) or IL-4 (2 µg/mL) for 2 hours at 37°C. HRP-conjugated avidin was added and incubated for 1 hour at 37°C. The assays were developed with DAB (Invitrogen) and stopped with tap water. Data were analyzed using ImmunoSpot software (CTL) and presented as the number of spot-forming cells (SFCs)/10⁶ PBMCs.