Progres c10plus

A morphological examination of immunostained tissue sections was carried out on a Zeiss Axioplan microscope equipped with a color change-couple device (CCD) camera (ProgRes C10plus, Jenoptik, Jena, Germany). Morphometric analysis was performed using KS 400 imaging software (Carl Zeiss Vision, Hallbergmoos, Germany), as described previously [13 (link)].

A polarizing light microscope (Nikon, Optiphot-2, Tokyo, Japan) equipped with a Linkam THMS 600 heating stage, a Linkam TP 91 control unit, and a Jenoptic ProgRes C10Plus camera with Jenoptik ProgRes CapturePro software was utilized for PLM observations. The samples were heated, melted, quenched, and captured during cold crystallization.

The pseudo-pregnant uteri (contains no conceptus/implantation sites; 7dpc) were collected and fixed in 4% formalin and paraffin embedded. For histology 3-5 µm thick sections were cut and stained with haematoxylin and eosin (H&E). The PAS stain allows the detection of polysaccharides such as glycogen. Tissue sections were deparaffinized and hydrated. Sections were then immersed in periodic acid solution (#11415, Morphisto, Frankfurt am Main, Germany) for 12 minutes at room temperature (RT), and then rinsed twice in distilled water. Slides were then immersed in Schiff’s reagent (#1.09033, Merck, Darmstadt, Germany) for 15 minutes at RT, followed by 10 min in running tap water. Finally, a counterstain with Hematoxylin solution was performed (4 minute incubation in Mayers Hematoxylin, 10 minutes washing in running tap water, dehydration and section mounting). The presence of glycogen with the Schiffs’ reagent gives a purple-magenta colour and hematoxylin staining gives the nuclei a blue colour. Images were taken using a digital camera ProgRes C10 plus (Jenoptik, Jena, Germany) mounted on an Zeiss Axio Imager 1 microscope (Zeiss, Oberkochen, Germany), with the ImageAccess Enterprise 6 Software (Imagic Bildverarbeitung, Glattbrugg, Switzerland). Appropriate positive and negative controls were used to confirm the adequacy of the staining.

For histology, tumors were fixed in 4.5% formalin (SAV LP GmbH, Flintsbach am Inn, Germany) or zinc (IHC Zinc-Fixative, BD Biosciences, Franklin Lakes, USA) and embedded in paraffin (Paraplast® Embedding Media, McCormick Scientific, Leica Microsystems, Wetzlar, Germany). Formalin-fixed sections were cut at 4 μm, deparaffinized and stained with either H&E or with the respective antibodies for immunohistochemistry. Slides were stained using an automatic immunostainer (Discovery XT, Ventana Medical Systems, Inc., Tucson, USA) according to the manufacturer’s standard protocols with antibodies against CD31 (Abcam Inc., Cambridge, USA), β₃-Integrin (Abcam Inc.), glucose transporter 1 (GLUT1, Abcam Inc.) and Ki67 (ThermoFisher Scientific, Waltham, USA). Images were acquired under a microscope (Axio Imager A1, Carl Zeiss AG, Oberkochen, Germany) using a coupled digital camera (ProgRes® C10plus, JenOptik, Jena, Germany) and the software ImageAccess (Version 6, Imagic Bildverarbeitungs AG, Glattbrugg, Switzerland) or extracted from digitized whole slide imaging (Nanozoomer, Hamamatsu) using the corresponding software.

Samples were fixed immediately in a solution of 5% formaldehyde in seawater. Prior to the histological procedures, the samples of P. tenacior, which contained siliceous spicules, were desilicified for 2 h in a solution of 5% hydrofluoric acid, while D. avara samples were decalcified for 2 h in a 5% solution of ethylene-diamine-tetracetic acid (EDTA) (Eerkes-Medrano & Leys, 2006 (link)) to remove the calcareous material included in its protein-made skeletal fibers (Galera et al., 2000 (link)).
Samples of both species were subsequently rinsed in distilled water, dehydrated through a graded ethanol series (70%, 96%, and absolute), rinsed in toluene/ethanol (1/1), and then in pure toluene, and embedded in paraffin for histological examination. Histological sections, 5 µm-thick, were obtained using an Autocut Reichert-Jung microtome 2040 (R. Jung GmbH, Nubloch, Germany). Sections were deparaffined with xylene, stained with hematoxylin and examined through a Zeiss Axioplan II compound microscope connected to a digital camera (Prog Res™ C 10^plus from JENOPTIK). Four sections were cut from each individual sponge, separated at intervals of ca. 1 mm to avoid cutting more than once the same reproductive structures.

Leaf silhouettes were prepared by sticking detached leaves on to a sheet of paper and scanning them using a Perfection V500 Photo scanner (Epson). For the developmental series, L6 leaves were dissected from the meristem under a stereomicroscope, mounted in water between a slide and coverslip, and imaged using an Axio Zoom.V16 microscope (Carl Zeiss Microscopy, Jena, Germany; http://www.zeiss.com/). For leaf shape analysis of in vitro grown plants, L5 leaves were collected, mounted in water between a slide and coverslip, and imaged using a stereomicroscope (SMZ 1000, Nikon) coupled to a camera (ProgResC10plus, Jenoptik).

Human HNC cell lines were plated at a density of 30,000 cells/mL on sterile round glass coverslips in a 12-well dish. The following day, the medium was replaced with a fresh serum-free medium, with or without 30 µg oxLDL. After 48 h of exposure, cell monolayers were fixed with 4% paraformaldehyde in Dulbecco’s Phosphate-Buffered Saline (DPBS, Lonza, Verviers, Belgium). Cells were rinsed with DPBS and colored with Oil Red O (Merk Sigma, Darmstadt, Germany) for 15 min at room temperature. Cells were rinsed three times with distilled water and the slides were mounted with aquatex^® (Merk Sigma, Darmstadt, Germany). The appearance of cells, with or without oxLDL, was documented by phase-contrast microscopy using a Zeiss axioplan microscope equipped with a color charge-coupled device (CCD) camera (ProgRes C10plus, Jenoptik, Jena, Germany).