Historesin embedding kit

For the histological procedure, five foragers bees were randomly collected from each experimental group and anesthetized at a cold temperature (4 °C) for one minute. Then, the bees’ parietal fat body was dissected with a digital stereo microscope (LEICA EZ4 HD) and immersed in a fixative solution (paraformaldehyde 4% in phosphate-buffered saline (PBS), 0.1 mol L¹, pH 7.4) for 24 h at 4 °C. Following the fixation period, these organs were immersed in PBS (pH 7.2–7.6 at 25 °C) for one hour and gradually dehydrated in ethanol, according to Silva-Zacarin et al. [42 ]. Upon these procedures, all organs were embedded in historesin (Historesin Embedding Kit, Leica Biosystems Nussloch GmbH, Heidelberger Str. 17-19) according to Leica Biosystems’ instructions. After complete polymerization of historesin, organs from five individuals from each group were fixed in wooden cubes (1 cm × 1 cm × 1 cm) and, using a microtome (LEICA RM2255), histological sections of 6 µm were performed gradually in the longitudinal direction of the organ. Histological sections were placed on microscopies slides, stained using the hematoxylin and eosin (HE) technique [43 ], and fixed to coverslips using dibutylphthalate polystyrene xylene (DPX) mounting medium for microscopic examination (Sigma-Aldrich, Saint Louis, MO, USA, 06522).

Samples were collected from in vitro-cultured seedlings and were fixed in Karnovsky solution (Karnovsky, 1965) for 24 h. After that, they were dehydrated in an increasing ethanol series (10–100%) and subsequently infiltrated with synthetic resin using a Historesin embedding kit (Leica, www.leica-microsystems.com), according to the manufacturer’s instructions. Tissue sections (5 μm) were obtained using a rotary microtome (Leica) and stained with toluidine blue 0.05% in a phosphate buffer and citric acid pH 4.0 (Sakai, 1973). Permanent slides were mounted with synthetic resin (EntellanR, Merck).

Small pieces of fresh fruit or leaf samples were fixed in 4% paraformaldehyde, dehydrated in a series of ethanol and later on embedded in a commercial resin (Leica Historesin Embedding Kit, Heidelberg, Germany). Samples were cross-sectioned into slices 4 µm thick using a Leica microtome (RM2125; Heidelberg, Germany). Three biological samples were analysed per species. Samples were inspected under a fluorescence microscope (Nikon, Eclipse E800, Tokyo, Japan) using a UV filter (excitation filter: 330–380 nm; dichroic mirror:400 nm; barrier filter: 420 nm) and microphotographs taken with a Nikon camera (DXM1200) coupled to the microscope.

The whole heads of two C. nicterus and one A. ocellifera were fixed in 4% paraformaldehyde in phosphate-buffered saline (PBS) 0.01 M for 4 h and rinsed overnight in PBS at 4 °C. Then we decalcified them by immersion in 7% EDTA for 24 h, rinsed in distilled water, dehydrated in an ascending ethanol series, and embedded in glycol-methacrylate resin (Historesin Embedding Kit, Leica, Wetzlar, DE) following the instructions of the manufacturer. Transverse sections of the head region containing the eyes were cut at 2 μm, mounted on clean glass slides, stained for 1 min with Azure II – Methylene Blue on a hot plate [64 (link)], mounted with Entellan (Merck, Darmstadt, DE), and photographed under a light microscope.