Leica historesin

Samples were taken from 0.5 cm above the tips of adventitious roots. The samples were fixed in FAA 50 (formaldehyde, acetic acid, 50% ethyl alcohol) [63 ] for 48 h and then stored in 70% alcohol. Afterward, they were dehydrated in ethanol series and embedded in methacrylate resin (Leica HistoResin, Leica, Wetzlar, Germany) [64 ]. The samples were sectioned on a semi-automatic rotary microtome and cross-sections (4-μm-thick) were stained with 0.05% Toluidine Blue pH 4.7 [65 (link)]. The slides were analyzed on a Leica DMR photomicroscope with DFC 425 camera (Leica, Wetzlar, Germany) attached. Quantitative analyses were performed using LAS software (V3.8 Leica, Wetzlar, Germany).

For the anatomical analyses, the material collected from the bracts (pre-anthesis), sepals (post-anthesis), labellum and column (pleuridia) were fixed in 1 % glutaraldehyde, 4 % formaldehyde and sodium phosphate buffer pH 7.2–0, 1 M (McDowell and Trump 1976 (link)), without vacuum, then dehydrated in an ascending ethanol series (10 %, 30 %, 50 %, 70 %, 90 % and 100 %) and embedded in hydroxyethyl methacrylate (Leica® historesin; Gerrits and Smid 1983 (link)). Serial cross-section and longitudinal sections approximately 3 μm thick were made using a rotary microtome (Leica Autocut) and stained in toluidine blue O (C.I. 52040) in 0.1 M sodium acetate buffer pH 4.7 (O’Brien et al. 1965 ). The permanent slides were mounted in Canadian balm and observed by light microscopy in brightfield (Leica DMR).

The fresh material was immediately fixed in formaldehyde, acetic acid, and 50% ethyl alcohol (1: 1: 18) and subsequently transferred to and stored in 70% ethanol (Johansen, 1940 ). Flower buds, flowers, and fruits at different stages of development removed from herbarium were rehydrated for 36 h in a 5% sodium hydroxide solution (Anderson, 1963 (link)), thoroughly washed with distilled water, submitted to an increasing ethanol series, and stored in 70% ethanol. For the anatomical study, the material was embedded in Leica historesin after dehydrating it in an ethyl alcohol series (Guerrits and Horobin, 1991 ).
The material was transversally and longitudinally sectioned at a thickness of about 8 μm with a rotary microtome, stained with toluidine blue in an acetate buffer, pH 4.7 (O’Brien et al., 1964 (link), modified), and mounted in Entelan^® synthetic resin.

Five larvae (L4; 24 h after exposure) and five pupae (one-day old; 96 h after exposure) of the same previous insecticidal treatments were collected and subjected to an assessment of cell death of muscular and nervous cells associated with the central nervous system, which is associated with swimming. The abdomens were dissected in insect physiological solution (0.1 M phosphate buffer under pH 7.4) and subsequently fixed for 24 h in 2.5% glutaraldehyde in 0.1 M cacodylate buffer (pH 7.4).
The fixed material was dehydrated in a crescent ethanol solution (70-100%) and embedded overnight in historesin Leica® (Heidelberg, Germany). The sample was subsequently embedded in historesin with hardener and subjected to microtomy. Five μm slices were transferred to glass slides and treated with proteinase K [10 μM/mL of Tris-HCl (10 mM, pH 7.4)] for 1 h at 37°C. The slides were washed three times in 0.1 M phosphate buffer (pH 7.4) and subsequently marked with the TUNEL reaction (Roche Aplied Science, Penzberg, Germany) for 1 h at 37°C. The slides were subsequently washed and covered with anti-fading media (Mowiol, Sigma-Aldrich Brasil, São Paulo, SP, Brazil). The slides exhibiting the cells under study were analyzed under a fluorescence microscope using a WU filter (BX60, Olympus, Center Valley, PA, USA).

Fixed stem segments were subsequently dehydrated in a graded ethanol series (50%, 70%, 80%, 90%, 95%, and 100%) for 1 h at each stage, and embedded in methyl methacrylate Historesin Leica^® (Leica Inc., Buffalo Grove, IL, USA) in a 3:1, 1:1, and 1:3 ratio, as well as in pure resin, according to the manufacturer’s instructions. Blocks were stored in polyethylene cases. After 2 days, the blocks were collected and sectioning was performed on a rotary microtome Leica RM2255 (Leica Inc., Buffalo Grove, IL, USA). The 5-µm sections were then dyed with toluidine blue in acetate buffer (pH 4.7). Images were using a Leica ICC50 camera with phase contrast. Anatomical data were quantified from the micrographic images using the Image-Pro Plus^® software (Media Cybernetics Inc., Rockville, MD, USA).