Lsm710 confocal laser scanning microscope

The primer pairs for the three CCR genes were used to amplify the cDNA fragments encoding the full-length CCR proteins. The PCR fragments for each CCR gene were inserted into the vector 1305GFP at the N-terminus of the green fluorescent protein (GFP) under the control of cauliflower mosaic virus 35S promoter. An Agrobacterium tumefaciens strain carrying the 35S::CCR1-GFP, 35S::CCR2-1-GFP, 35S::CCR2-2-GFP or 35S::GFP plasmid was infiltrated into Nicotiana benthaminana leaves and analyzed with confocal microscopy 48 h after agroinfiltration as described previously (Goodin et al., 2002 (link)). Fluorescence of GFP was observed with a Leica LSM710 confocal laser scanning microscope.

Zebrafish hearts were removed from culture at day 0, 1, 3 and 5 before being fixed, embedded and sectioned as described above. 20 images at high magnification were acquired for each zebrafish heart at each time point using a Zeiss 710 confocal laser scanning microscope. DAPI positive nuclei were counted using ImageJ and compared to TUNEL positive nuclei. Four heart sections were analysed in each heart for quantification of TUNEL staining. TUNEL assay (DeadEnd™ TUNEL assay) was performed according to the suppliers protocol (Promega UK, Southampton, UK). Fluorescence microscopy was performed using a Leica LSM 710 confocal laser scanning microscope and Leica ZEN imaging software. Histological examination was performed using an Olympus BX51TF (Olympus, Japan) brightfield microscope linked to a video camera (micropublisher 3.3 RTV QImaging, Canada) and analysed using commercial software (Qcapture Pro 6.0 imaging software, QImaging).

Third instar larval wing imaginal discs or pupal retinas were dissected in PBS and fixed with 4% formaldehyde in PBS at room temperature for 20 min. After fixation, the tissue was washed twice with PBT, permeabilised with PBS containing 0.3% (v/v) Triton X-100 (PBT) for 30 min at room temperature, blocked with PBT containing 10% normal goat serum (NGS) for 30 min at room temperature and incubated at 4 °C overnight in primary antibody. Following five washes in PBT, the tissue was incubated for 1 h at room temperature in secondary antibody, then washed and mounted in Vectashiled with DAPI (Vector) on glass slides. Primary antibodies used: rat anti-E-cadherin (DCAD2), 1/100 (Developmental Studies Hybridoma Bank), rat anti-ASPP, 1/500^{31 (link)}, mouse anti-β-Galactosidase, 1/500 (Promega). Images were acquired on a Zeiss LSM 710 confocal laser-scanning microscope or a SP5 laser scanning confocal (Leica). Images were processed with ImageJ.