Axiocam icc3 digital camera

Morphometric and immunohistochemical analyses were performed in the four WAT depots from 14-month-old rats. Fragments of tissues were fixed in 4% paraformaldehyde in 0.1M phosphate buffer pH 7.3 overnight at 4°C, and then washed in the same buffer. For paraffin embedding, samples were dehydrated in ethanol, cleared in xylene, and embedded in paraffin blocks at 60°C.
For immunohistochemistry analysis, 5 μm sections were immunostained by means of the avidin-biotin technique using a commercial anti-MAC2 antibody (1:350 in PBS, Cederlane, Hornby, Ontario, Canada), counterstained with hematoxylin and mounted in Eukitt (Kindler, Freiburg, Germany). Images were acquired with a Zeiss Axioskop 2 microscope equipped with AxioCam ICc3 digital camera and AxioVision 40V 4.6.3.0 Software (Carl Zeiss, S.A., Barcelona, Spain). The software was also used to measure the diameter of 100 adipocytes (from one random field) in two non-consecutive hematoxylin/eosin stained sections. Image analysis from all groups was examined in a blind fashion.

Fresh young panicles were fixed in ethanol:acetic acid (3:1, v/v) in three rounds of 10 min under vacuum kept on ice (450 mmHg, vaccum pump R-400, Pobel, Madrid, Spain). Subsequently, the fixed panicles were stored at 4°C for up to 3 months.
Ethanol and acetic acid were used as fixatives because they have been previously proved to be successfully employed in proteomic analysis (Ahram et al., 2003 (link); De Souza et al., 2004 (link); Milcheva et al., 2013 (link)) (Figure 1). Although the anther length is generally used as a criterion to determine the developmental stages of anthers in rice (Kerim et al., 2003 (link); Itoh et al., 2005 (link); Nonomura et al., 2011 (link)), an anther from each fixed flower was removed using fine forceps under a dissection microscope (Stemi 2000-C stereomicroscope, Carl Zeiss, Göttingen, Germany) equipped with a cold-light source to stage meiosis as much accurately as possible. Anthers were then stained in acetocarmine solution, squashed on ethanol-cleaned slides and checked under a PrimoStar light microscope (Carl Zeiss, Göttingen, Germany). The identification of the meiotic developmental stage was based on previous cytological descriptions of rice male meiosis (Chen et al., 2005 (link); Itoh et al., 2005 (link)). Photographs were taken using an AxioCam ICc3 digital camera (Carl Zeiss, Göttingen, Germany) attached to the microscope.

The influence of Ascaris ESP on the morphology of biofilms was assessed using the macrocolony biofilm model (Serra and Hengge, 2017 (link)). Experiments were carried out using the same strains as for the crystal violet biofilm formation assay. Cells were grown overnight in salt-free LB medium at 37°C. 5 μL of the overnight culture was spotted on salt-free LB agar plates containing Congo red 40 μg/mL and Coomassie brilliant blue 20 μg/mL. 35 mm petri dishes (Sarstedt, Nümbrecht, Germany) were used to grow one colony per plate. After autoclaving and cooling to 42°C, agar was prepared with controls and treatments at the indicated final concentrations. Colonies were incubated at 28°C for up to 5 days. Macrocolonies were visualized at 10X magnification with a Stemi 2000-C stereomicroscope (Zeiss, Oberkochen, Germany) and photographed with an AxioCamICC3 digital camera (Zeiss).