Tcs spii

Five-micron thick paraffin sections were washed in phosphate-buffered saline and processed for staining with anti-Edn2, anti-glutamine synthetase (GS) and anti-glial fibrillary acidic protein (GFAP) antibodies as previously described [16 (link)]. All sections were counterstained with 4’-6’diamidino-2-phenylindole (DAPI; Roche) prior to confocal microscopy (Leica TCS SP II, Leica Microsystems GmbH, Wetzlar, Germany) at the Centre for Microscopy and Cellular Analysis, The University of Western Australia.

The coding sequence (CDS) of SvMBD5 without the terminator codon was cloned into the pEarleyGate101 vector fused with YFP (yellow fluorescent protein) using Gateway Technology (Life Technologies, Carlsbad, CA, USA) (Figure 1). The Gateway primers for the subcellular localization of SvMBD5 were as follows: Forward: 5’-GGGGACAACTTTGTACAAAAAAGTTGGAATGTCATTGTCAGCAACTCC and Reverse: 3’-GGCGGCCGCACAACTTTGTACAAGAAAGTTGGGTAACGCTTTCTGTTACCAT. The constructs were transiently expressed in Arabidopsis protoplasts as described by Miao et al. [39 (link)]. YFP fluorescence was imaged under a confocal laser-scanning microscope (Leica TCS SPII, Leica Microsystems, Wetzlar, Germany).

Fluorescent images were taken using the confocal microscopes Leica TCS SP5 and Leica TCS SP-II. Image analysis was performed using the Fiji ImageJ software and Adobe Photoshop. To quantify the percentage of Col XII in the post injured myocardium, the area positive for Col XII expression was divided by the whole post-injury myocardium area, but excluding the epicardium. N represents a number of hearts. In order to obtain representative data, at least 2 sections of each heart at different time points were imaged and analyzed. Statistics were calculated with the Prism GraphPad software. All results are expressed as the mean ± standard error of the mean (S.E.M.). Bright-field images were taken using a Zeiss Axioplan 2 microscope coupled to an AxioCam Color camera.

Cells were fixed with 1% formaldehyde in PBS and permeabilized with 0.01% saponin in PBS. Rabbit polyclonal antibodies against human cathepsin D were used, followed by Alexa 488-conjugated goat anti-rabbit IgG (Invitrogen) staining. Cells were observed using a laser scanning confocal microscope (Leica TCS SPII). DAPI (Invitrogen) was used for nuclear staining.

After the TetOff induction, cells grown on Lab-Tek chambered cover glass were treated with L-Hcy (100 µM) for the last 24 h of the TetOff induction period. Then, cells were fixed with 4% paraformaldehyde/0.1 M phosphate buffer (PB). We rinsed the cells with PBS, and permeabilization was performed using 0.25% Triton X-100/PBS. Next, we blocked the cells in blocking solution (3% goat serum and 1% bovine serum albumin in PBS) and incubated in P44, CP13, PS199/202, TauC3, or TOC1 antibody solution, followed by Alexa 488 anti-mouse IgG and Alexa 594 anti-rabbit IgG, or Alexa 488 goat-mouse IgM in blocking solution (Molecular Probes, Eugene, OR, USA). A confocal laser microscope (version 09-2004, TCSSPII; Leica, Heidelberg, Germany) was used to image the cells. The number of cells positive for each tau antibody was calculated in 4 randomly chosen visual fields per well and each experimental group was repeated with 4 wells.

Fluorescence microscopy was used to identify the cellular localisation of the contrast agents. Cells were grown and labelled in culture slides (BD Biosciences) and the same immunostaining procedures as described for flow cytometry were applied. For imaging the lysosomes, a rabbit antibody against the Lysosomal-Associated Membrane Protein 2 (LAMP2, Abcam, Cambridge, England) was used in conjunction with secondary goat anti-rabbit antibody conjugated to Alexa-Fluor-488 (Life Technologies, Paisley, Scotland). Particles and lysosomes were imaged using confocal microscopy (Leica TCS SP II) using a pinhole size of 1 airy unit. Nuclei were stained using 4′,6-diamidino-2-phenylindole (DAPI, Life Technologies) and imaged using wide-field fluorescence.
For transmission electron microscopy (TEM) cells were grown and labelled in 3.5 cm dishes, followed by fixation with 4% formaldehyde/2.5% glutaraldehyde, post fixation with 1% osmium tetroxide, dehydration and embedding in epoxy resin. Thin sections (70 nm) were then collected over copper grids containing a formvar support film. For analysis of the contrast agents, those were simply dispersed of over the grids containing the support film and allowed to dry. Samples were analysed with a FEI Technai G2 Spirit BioTwin microscope operated at 100 kV.