Lsm 510 meta confocal fluorescence microscope

A full-length UvCdc10 or MoSep4 gene–coding region and 2 kb native promoter were cloned into vector p3300neo-GFP. The EHA105 strain with the p3300neo-UvCdc10-GFP and p3300neo-MoSep4-GFP vector was transformed using A. tumefaciens-mediated transformation by coculture with conidia of the ΔUvCdc10 and ΔMoSep4 mutants, respectively. Subcellular localization of UvCdc10-GFP or MoSep4-GFP was photoed using a Zeiss LSM 510 Meta confocal fluorescence microscope, with the combinations of excitation and emission wavelengths of 488 nm/bandpass 500 to 550 nm for GFP.

Serum samples of 28–33 week-old mice (diluted 1:100 in PBS) were incubated on HEp-2 slides (Bio-Rad Laboratories) for 60 min, as previously described (8 (link)). After washing with PBS, slides were incubated with Alexa Fluor-488 conjugated donkey anti-mouse IgM or IgG F(ab′)₂ fragments (Jackson ImmunoResearch) for 1 h. After washing and staining for 5 min with DAPI, slides were embedded in VectaShield (Vector Laboratories). The LSM 510 META confocal fluorescence microscope (Zeiss) was used to measure fluorescence intensity.

Cells were grown on to glass cover slips and fixed with 2% paraformaldehyde in PBS. The fixed cells were rinsed with PBS, permeabilized with 0.2% Triton X-100 in PBS for 5 min, and then blocked with 3% BSA in PBS for 1 h at room temperature. Subsequently, the cells were incubated with indicated primary antibodies or non-specific IgG overnight at 4 ˚C, washed in PBS, and then exposed to Alexa 568-conjugated secondary IgG for 1 h at room temperature, respectively. The cells were stained with Hoechst 33342 before the glass cover slips were washed in PBS and mounted on glass slides. The cells were examined under the Zeiss LSM 510-Meta confocal fluorescence microscope (Carl Zeiss, Jena, Germany).

Paraffin sections were dewaxed, and rehydrated using xylene and successive ethanol baths. After extensive washes in phosphate-buffered saline (PBS), tissue sections were incubated with specific primary antibodies for 1 h at 37°C in a humidified atmosphere and then washed three times with PBS. For immunohistochemical studies, the following antibodies were used: ɑ-smooth muscle actin (ɑ-SMA) (1:100), neutrophil (1: 50), CK19 (1: 100), F4/80 (1: 75), HIF-1ɑ (1: 50), vWF (1: 100), VEGFR1 (1: 100). Samples were incubated with peroxidase- or fluorescently-compound-conjugated secondary antibodies for 1 h at 37°C followed by extensive washes in PBS. Fluorescent images were visualised using a Zeiss LSM 510 META confocal fluorescence microscope (Carl Zeiss, Jena, Germany). The number of activated HSCs in the liver was estimated using double immunofluorescence staining with the anti-ɑ-SMA and anti-desmin antibodies. Nuclear staining (blue fluorescence) was obtained by treating liver sections with 4,6-diamidino-2-phenylindole (DAPI)