Casein blocking buffer

For the immunohistochemical analyses, the material was fixed in 4% formaldehyde. Samples were dehydrated in series of ethanol and HistoChoice® Clearing Agent (Sigma-Aldrich) and embedded in paraplast. The paraplast blocks were cut into 5-μm-thick sections. Slide-mounted sections were deparaffinized (dewaxed) in HistoChoice® Clearing Agent (Sigma-Aldrich), rehydrated gradually through a series of ethanol dilutions and rinsed in water. Blocking of non-specific binding sites was performed with casein blocking buffer (Thermo Fisher) overnight at 4 °C prior to the incubation with anti-HC1 and HC2 antibodies (the antibodies were raised against the hemocyanin subunits of the Dubia roach, Blabtica dubia; a generous gift from Prof. T. Burmester, Hamburg University, Germany) diluted 1:500. In parallel performed control experiments, the primary antibody was omitted. After overnight incubation at 4 °C in a humidified chamber, sections were washed with PBS with 0.1% Triton X-100 and 0.05% Tween 20 and incubated with Alexa Fluor 488 or Cy3 goat anti-rabbit secondary antibodies (Life Technologies) for 4 h at room temperature. After rinsing with PBS, the sections were mounted in ProLong Gold antifade reagents with DAPI (Invitrogen) and analyzed in the DMR Leica epifluorescence microscope equipped with appropriate filters. Each staining experiment was performed in triplicate.

Half-well 96-well plates were coated overnight at RT with Galanthus nivalis lectin (Vector laboratories) at 20 µg/mL in 0.1 M NaHCO₃ pH 8.6. The next day plates were washed and Casein blocking buffer (ThermoScientific) was added. After 30 min a 2 µg/mL concentration of untagged 16055 SOSIP.v8.3 in Casein was added for 2 h at RT. Plates were then washed and a 1:100 dilution of rabbit sera in TBS/2% skimmed milk/20% sheep serum was added for 30 min. Next, mAbs were added for 1.5 h at RT at a final concentration that gave 80% of maximal binding signal as determined by an ELISA in the absence of serum (D11A.F9: 0.1 µg/mL; D11A.F2: 0.1 µg/mL; RM19R: 2 µg/mL, in TBS/2% skimmed milk/20% sheep serum). Plates were then washed three times and a 1:3000 dilution of HRP-labeled donkey anti-human IgG (Jackson Immunoresearch) in Casein was added for 1 h at RT. Washing and colorimetric detection then proceeded as explained for Serum antibody ELISA above. Residual binding was determined as described previously^{27 (link)}.

Purified Strep-II-tagged sE2 monomer (1.0 µg ml⁻¹ in TBS) was coated for 2 h at room temperature on 96-well Strep-TactinXT coated microplates (IBA LifeSciences). Plates were washed with TBS twice before incubating with serially diluted mAbs and CD81-LEL-hFc (R and D systems) in casein blocking buffer (Thermo Fisher Scientific) for 90 min. After three washes with TBS, a 1 : 3000 dilution of HRP-labelled goat anti-human IgG (Jackson Immunoresearch) in casein blocking buffer was added for 45 min. After washing the plates five times with TBS +0.05 % Tween-20, plates were developed by adding develop solution [1 % 3,3′,5,5′-tetraethylbenzidine (Sigma-Aldrich), 0.01 % H₂O₂, 100 mM sodium acetate, 100 mM citric acid] and the reaction was stopped after 3 min by adding 0.8 M H₂SO₄. Absorbance was measured at 450 nm and data was visualized and analysed on GraphPad Prism.