Polybed 812

Adult females of the golden orb-web spider, Nephila clavata (Araneae: Nephilidae) were collected in a local area near the Cheonan campus of Dankook University, Chungnam, Korea. All spiders were maintained under ambient conditions with natural lighting in enclosures comprising a wooden frame (height × length × width = 50 × 50 × 10 cm) with glass panels on the front and back, and fed insect larvae and water daily.
For histologic preparation, specimens were anesthetized with CO₂ and dissected under a dissecting light microscope in a drop of spider Ringer's solution consisting of 160 mM NaCl, 7.5 mM KCl, 4 mM CaCl₂, 1 mM MgCl₂, 4 mM NaHCO₃, 20 mM glucose, pH 7.4 (Moon and Tillinghast 2013 (link)). Both of spinnerets and silk glands were gently removed and fixed in alcoholic Bouin’s solution, and dehydrated through an ethanol series from 30% to l00% (30 min at each concentration, with one repeat at 100% ethanol). After dehydration, the specimens were transferred to xylene for clearing at room temperature to 60°C, and they were embedded with Paraplast embedding medium (Fisher Scientific Co., Pittsburgh, Pa, USA) at 60°C. The sections were cut with a thickness of approximately 5 µm using a rotary microtome, Histocut 820-II (Reichert-Jung, Germany) and they were stained with hematoxylin and eosin (H & E) solutions.
For scanning electron microscopic (SEM) experiment, the specimens were prefixed in a mixture of 2% paraformaldehyde and 2.5% glutaraldehyde buffered with 0.1 M phosphate buffer at pH 7.4. Postfixation was performed with 1% osmium tetroxide in the same buffer and washed several times in 0.1 M phosphate buffer. Following fixation, the specimens were carefully dehydrated in ascending concentrations of ethanol from 30% to l00%, and then either critical point-dried or transferred to hexamethyldisilazane (HMDS) for air-dry. All samples were coated to a thickness of approximately 20 nm with gold-palladium alloy using a sputter coater and examined on a Hitachi S-4300 (Hitachi Co., Japan) field emission scanning electron microscopy (FESEM) operated with an accelerating voltage of 5–15 kV.
For transmission electron microscopic (TEM) experiment, both spinnerets and silk glands were fixed and dehydrated according to the same protocol for SEM experiment. The specimens were then embedded in Poly/Bed 812-Araldite medium (Polysciences Inc., Warrington, PA, USA) via propylene oxide. Semi-thin sections, 0.5–1.0 µm thick, were obtained using an LKB Ultratome V (LKB, Stockholm, Sweden) and were stained with 1% toluidine blue (dissolved in 1% borax). Microscopic images were photographed using Zeiss Axiophot microscope (Carl Zeiss, Jena, Germany) coupled with a Motic digital imaging system (Motic Instruments Inc., Richmond, Canada). Ultrathin sections were obtained using an Ultra 45° diamond knife (Diatome, Hartfield, PA, USA), and were double stained with uranyl acetate, followed by lead citrate. After these treatments, the sections were examined with a JEM 100 CX-II transmission electron microscope (JEOL Ltd., Tokyo, Japan) at 80 kV.

Needle samples # 45 and # 48 were collected and subjected to 37°C and 43°C for 24 hours, and samples at 25°C were collected as controls. After treatment, three mature needles were sampled. The needles were cut into a 3 mm segment size with a blade and placed in a 5 ml penicillin bottle; 2 ml of 3% glutaraldehyde solution was added, the air in the bottle was extracted with a syringe, which caused the needles to sink entirely to the bottom of the bottle. Samples were then stored in a 4°C refrigerator for 24 h. The specimens were rinsed with 0.1 mol L^-1 phosphate buffer solution with a pH of 7.0 3 times for 30 minutes each time. The specimens were subsequently dehydratedin a series of graded alcohol solutions, namely, 15%, 30%, 50%, 70%, 80%, 90%, 95%, 100% solution, followed by acetone solution at room temperature. The samples were infiltrated and embedded with Poly Bed 812 epoxy resin, dried and stored for 8 hours. The samples were then sectioned using ultratome III ultramicrotome (LKB, Bromma, Stockholm, Sweden). The samples were subsequently stained in uranium dioxide acetate and lead citrate and then observed via the transmission electron microscope (TEM) (JEM-2100, JEOL Ltd., Tokyo, Japan). From the TEM images, ten chloroplasts were selected for observation. The shape of the chloroplast is approximated to be an ellipse, and the calculation formula of ellipse area is as follows: S=πab, where S is the elliptical area (µm²), a is the semimajor axis (µm), and b is the semiminor axis (µm).

For TEM analysis, HIV-1-infected PBMCs treated or not with 100 ng/mL of IL-27 during 48 h were fixed in 2.5% glutaraldehyde in 0.1 M cacodylate buffer (pH 7.2) for 1 h at 25°C and postfixed with a solution of 1% OsO₄ containing 0.8% potassium ferricyanide and 2.5 mM CaCl₂ in the same buffer for 20 min at 25°C. Then, the samples were dehydrated in an ascending acetone series and embedded in Polybed 812 resin (Polysciences, USA). Ultrathin sections were obtained, stained with uranyl acetate and lead citrate, and examined with the transmission electron microscope JEM 1011 (Jeol, Tokyo, Japan) at the Fiocruz Electronic Microscopy Platform.