Paraplast

In situ hybridization experiments were performed as previously described (Lee et al., 2003), with minor modifications. Briefly, wild type rice plants Oryza sativa L. ssp. japonica cv. Ilmi were grown on MS media for 6 days and stem sections were fixed in a solution containing 50% ethanol, 5% acetic acid and 3.7% formaldehyde. They were then embedded in paraplast (Sigma, St Louis,MO) and 8 μm cross‐sectional cuts were made using a microtome. Digoxigenin (DIG)‐labelled OsUPS1 antisense and sense probes were generated through in vitro transcription from the 5′UTR to the coding region (1.4 kb) with DIG‐labelled UTP (Roche, Mannheim, Germany) using the SP6 RNA polymerase and the T7 RNA polymerase, respectively (Roche, Mannheim, Germany). paraplast were removed and the sections were then dried on a slide warmer and incubated in a humidified box containing one of the probes (0.8 μg/slide) for 12 h. After hybridization, sections were washed and colour‐reacted in a solution containing blue tetrazolium chloride (NBT), 5‐bromo‐4‐chloro‐3‐indolyl‐phosphate (BCIP) and levamisole for 36 h in a dark humidified box and mounted with Permount^® (Fisher Scientific, Fair Lawn, NJ). Photographs were taken under a bright field illumination using a microscope (ZEISS AXIO Imager.A2, Germany) equipped with a camera (ZEISS Axiocam 506 Color, Germany).

After euthanasia of the animals, one knee joint was randomly obtained from each animal. For preparation of coronal sections, joints were flexed at 120 degrees and fixed in 10% buffered formalin (1.27 mol/L formaldehyde in 0.1 M phosphate buffer pH 7.2) for 48 h, then decalcified in 10% buffered EDTA for 90 days. Subsequently, the samples were dehydrated in ascending alcohols, rinsed in xylene, and embedded in Paraplast (Fisher Scientific, Waltham, MA, USA). Using a Leica^® RM 2255 microtome, 5 µm thick sections were cut at 200 µm intervals and mounted on slides (Superfrost plus) [73 (link)]. To optimally evaluate the joint, successive sections of the deepest planes of the joint were stained with toluidine blue, visualized with an optical microscope (Leica^® DM 2000 LED), and photographed with a Leica^® MC 170 HD digital camera. Then, only one slice per joint was selected, considering the plane of the blockage that crosses the lesion to the greatest extent and that presents the most pronounced alterations [48 (link)]. These sections were visualized and scanned using the TissueFAXS i PLUS Cytometer TissueGnostics Axio Observer 7 Carl Zeiss GmbH System (TissueGnostics GmbH, Vienna, Austria).

Limb tissues were treated as described previously [45 (link)]. In brief, they were fixed in Bouin's solution for 48 h before washing in 50% alcohol to remove the picric acid. They were then decalcified for 3 weeks in Calci-Clear (National Diagnostics, Atlanta, GA, USA) at room temperature. The limbs were dehydrated in a graded series of alcohols up to 100% (1 hr each), followed by two changes of Histoclear (Fisher Scientific, Houston) for 1 h each. They were then infiltrated overnight with Pararaplast (Fisher Healthcare, A Fisher Scientific Company, Houston, TX, USA), followed by a second overnight infiltration with fresh paraplast. After embedding in a third change of paraplast, longitudinal sections were cut at 10 μm, processed for staining with hematoxylin and eosin or Mallory’s trichrome, and photographed at 10 × magnification on a Leica microscope.

Inflorescences containing flowers at several developmental stages were collected at the Instituto de Biociências of Universidade de São Paulo and at the Instituto de Botânica in São Paulo. Vouchers of the species were provided and the vouchers were deposited in the herbarium of the Universidade de São Paulo (SPF). The material was fixed in formalin, acetic acid, 50% ethyl alcohol (FAA) for 24 h [16 ] or by buffered neutral formalin (BNF) for 48 h [17 ], and then stored in 70% ethyl alcohol.
Inflorescence meristems, flower buds, and anthetic and post-anthetic flowers were isolated, dehydrated in a butyl series [16 ], embedded in Paraplast (Fisher Healthcare, Houston, Texas, USA), and transversely and longitudinally sectioned using a Leica RM2145 rotary microtome (Leica Microsystems, Wetzlar, Germany). Serial sections around 12 μm thick were stained with astra blue and safranin [18 ], and mounted in Permount resin (Fisher Scientific, Pittsburgh, Pennsylvania, USA). Samples were observed and photographed using a Leica DMBL light microscope (Leica Microsystems, Wetzlar, Germany).