A3562

Around 10–15 nuclear subfractions from each of the control, early, and late preeclamptic placenta groups were pooled randomly within each group. The same procedures were used for the cytoplasmic subfraction and for total homogenates. The protein concentration was measured by fluorescence. The samples were loaded onto a 10% SDS-PAGE gel and separated under reducing conditions. Following this, they were transferred to a PVDF microporous membrane (Merck Millipore, Billerica, MA). In the next step, the membrane was incubated with 5% nonfat milk in TBST buffer (250 mM Tris-HCL pH 7.6, 1.5 M NaCl, 0.01% Tween) for one hour at room temperature, and then overnight at 4°C with a primary antibody diluted in 1% nonfat milk in TBST buffer: 1 : 500 for IKBKG (MA5-15155, Thermo Scientific), 1 : 500 for Lamin A/C (MA3-1000, Thermo Scientific), and 1 : 1000 for GAPDH (MA5-15738, Thermo Scientific). Following incubation, the membrane was washed in TBST buffer, incubated for one hour at room temperature with labeled alkaline phosphatase (A3562, Sigma-Aldrich; dilution 1 : 10000 in 1% nonfat milk in TBST) secondary antibody, and again washed. The analysis was then performed using an ECL detection kit according to the manufacturer's instructions. The optical density of bands was analyzed using the ImageJ software [26 ].

A volume of 10 µL of both saliva and salivary gland extracts, as well as 1.5 µg of recombinant IrSPI protein as positive control, were analyzed on 4–15% acrylamide gels under reducing conditions (Mini protean TGX stain-free gels, Bio-Rad, Hercules, CA, USA) followed by UV detection (ChemiDoc, Bio-Rad, Hercules, CA, USA), or were immunoblotted with sera from mice or rabbits after IrSPI immunization or tick infestation, respectively. Transfer to nitrocellulose membranes was performed using the transblot turbo (Bio-Rad) followed by saturation in TBS-5% milk for 1 h at RT and incubation for 1 h at RT with primary antibodies diluted in TBS-5% milk (1/1000 dilution for mice immune serum and 1/100 for rabbit serum). After three washes in TBS-5% milk, antibody binding was detected with the BCIP/NBT Color development system (Roche), using either anti-mouse or anti-rabbit alkaline phosphatase-conjugated antibodies (A3562 or A3687, respectively; Sigma-Aldrich) diluted 1:7500 in TBS-5%. Results were analyzed with the ChemiDoc instrument (Bio-Rad) and the accompanying ImageLab 1.5 software (Bio-Rad).

Isolated proteins from the tissue samples were coated overnight at 4 °C onto NUNC Maxisorp enzyme-linked immunosorbent assay (ELISA) plates at 2 µg/mL and 100 µL/well. The coating buffer was 0.1 M NaHCO₃, pH 9. The following day, the wells were washed with PBS supplemented with 0.01% Tween, and the residual binding sites were blocked with 5% PBS-milk. The wells were washed again, and 100 µL primary antibodies (1:2000; same as those used during the immunoblotting) were put into each well. After 1 h incubation at room temperature, the wells were washed again and secondary anti-mouse (A3562, Sigma Aldrich) and anti-rabbit (A3687, Sigma Aldrich) IgG whole molecule alkaline phosphatase antibodies produced in goat were prepared at 1:2000 dilution in 1% PBS-milk, and were applied (100 µL/well). After 1 h incubation at room temperature, the wells were washed again, and 100 µL/well alkaline phosphatase substrate (P4744; Sigma Aldrich) was added. Signals were measured using a microplate reader (Synergy H4 Hybrid; BioTek, Winooski, VT, USA) at 405 nm.

Enucleated eyes from two female patients (88 and 61 years old) with severe ischemic CRVO followed by neovascular glaucoma were investigated for expression of the proteins that showed higher concentrations in the vitreous than in blood. Sections (5 μm) of paraffin embedded eyes were dewaxed and demasked for 20 min in 100 mM sodium citrate, pH 6.0, in a steamer. After transfer to TBST (50 mM Tris / HCl pH 7.6, 0.9% NaCl, 0.02% Tween 20), sections were blocked with Ultra V Block (Lab Vision at medac GmbH, Wedel, Germany) and incubated with antibodies as listed in Table 2 for 3 h. After washing in TBST, an AP-labelled goat anti-mouse secondary antibody (A3562, Sigma-Aldrich, Taufkirchen, Germany) was applied for 1 h, or a biotin-labelled goat anti-rabbit secondary antibody (71-00-30, KPL, Gaithersburg, MD, USA) was applied for 1 h followed by streptavidin-coupled AP (71-00-45, KPL) for 1 h. Slides were washed and AP was made visible by the Vector Red AP Substrate Kit I (SK-5100, Vector Labs at Axxora, Lörrach, Germany). Sections were counter-stained with hematoxylin.