10 nm colloidal gold granules

Male or female antennae of two- or three-day-old adult wasps were fixed in a mixture of paraformaldehyde (4%) and glutaraldehyde (2%) in 0.1 M PBS (pH 7.4) at room temperature for 24 h, dehydrated in an ethanol series, and embedded in LR white resin (Taab, Aldermaston, Berks, UK) for polymerization at 60°C. Ultrathin sections (60–80 nm) were cut using a diamond knife on a Reichert Ultracut ultramicrotome (Reichert Company, Vienna, Austria). For immunocytochemical assay, the grids were subsequently floated in 25 μl droplets of PBSG (PBS containing 50 mM glycine) and PBGT (PBS containing 0.2% gelatin, 1% bovine serum albumin, and 0.02% Tween-20) and incubated with purified rabbit anti-MmedCSP3 antiserum (dilution 1: 2,000) at 4°C overnight. After washing six times with PBGT, the sections were incubated with secondary antibody (anti-rabbit IgG) coupled with 10 nm colloidal gold granules (Sigma, St. Louis, MO, USA) at a dilution of 1: 20 at room temperature for 90 min. Before being observed with a Hitachi H-7500 TEM (Hitachi Ltd., Tokyo, Japan), the sections were subjected to optional silver intensification for 15 min and stained with 2% uranyl acetate to increase the contrast. The serum supernatant from an un-injected healthy rabbit at the same dilution rate acted as the negative control. Three male and female adult antennae were respectively used in immunocytochemical assays.

The foreleg tarsus of female adult, and intact antennae detached from male and female adults were prefixed in a mixture of paraformaldehyde (4%) and glutaraldehyde (2%) in 0.1 M PBS (pH 7.4) for 24 h at room temperature, dehydrated in an ethanol series, and then embedded in Luria-Bertani white resin (Taab, Aldermaston, United Kingdom) for polymerization at 60°C. Ultrathin sections (60 nm) were cut by a diamond knife on a Reichert Ultracut ultramicrotome (Reichert Co., Vienna, Austria). For immunostaining, the grids were floated in droplets of PBS (containing 50 mM glycine), followed by PBGT (PBS containing 0.2% gelatine, 1% bovine serum albumin, and 0.02% Tween-20). The grids were then incubated with EoblOBP6 antiserum (diluted at 1:3000) at 4°C overnight. After rinsing six times in PBGT, the grids were incubated with secondary antibody (anti-rabbit IgG) coupled with 10 nm colloidal gold granules (Sigma) (diluted at 1:20) for 90 min at room temperature. The grids were then transferred to silver intensification and stained with 2% uranyl acetate to increase the contrast. Finally, sections were observed with HITACHI H-7500 TEM (Hitachi Ltd). The serum supernatant from an uninjected rabbit was used as the negative control.

Male and female antennae from 5-day-old M. alternatus adults were cut into small pieces and fixed overnight in 0.1 M PBS containing 4% paraformaldehyde and 2.5% glutaraldehyde at pH 7.4 and 4°C. Samples were dehydrated using an ethanol series and embedded in LR White resin (Taab, Aldermaston, Berks, United Kingdom). Ultrathin sections were cut with an LKB V Ultramicrotome (LKB Company, Bromma, Sweden) and mounted on formvar-coated 200 mesh grids. The sections were washed three times with 0.1 M PBS containing 50 mM glycine (buffer G) at pH 7.4, and blocked for 2 h at room temperature in 0.1 M PBS containing 2% gelatin, 5% bovine serum albumin, and 0.2% Tween-20 (buffer T) at pH 7.4. The sections were then stained overnight at 4°C with primary antibodies (dilution 1:4000) and subsequently stained with secondary antibodies (dilution 1:4000), which were coupled with 10-nm colloidal gold granules (Sigma, St Louis, MO, United States). After two washes in buffer T, buffer G, and water, the sections were finally examined with an H-7650 (Hitachi, Japan) transmission electron microscope. The preimmune serum from an uninjected healthy rabbit was used as a negative control.