Tetramethylrhodamine isothiocyanate tritc

Immunofluorescence was performed using the method reported in Rutkowski et al (2012) (28). Briefly, 1 × 10⁵ cells were seeded onto cover slips and allowed to attach for 24 h before being fixed using 4% paraformaldehyde and permeabilised using 0.3% Triton X-100. Non-specific binding was blocked using 10% goat serum before addition of antibodies overnight at 4°C. After washing, bound primary antibody was detected using a secondary antibody conjugated to a fluorescent tag. In some experiments phalloidin conjugated to tetramethyl rhodamine isothiocyanate (TRITC) (Sigma, St. Louis, MO) was used to stain F-actin. Cover slips were mounted on glass slides using Prolong Gold Antifade with 4,6-diamidino-2-phenylindole DAPI (Life Technologies) and cells were visualised using a Leica SP5 spectral scanning confocal microscope. To identify EphB4 specific staining, control samples included one to which an irrelevant mouse IgG was added, another to which the secondary antibody alone was added and a third to which no antibody was added.

TRIM probes directly labelled by PCR with either biotin-16-dUTP (20 µM, Roche Applied Science) or digoxigenin-11-dUTP (5 µM, 10x DIG Labeling Mix, Roche Applied Science) were hybridized with mitotic metaphase chromosomes and synaptonemal complexes according to previously published methods^{27 ,42} . Chromosome preparations, pre-treated with RNase and pepsin and post-fixed with formaldehyde, were denaturated for 2 min at 70 °C (mitotic chromosomes) or 80 °C (meiotic chromosomes) and hybridized overnight at 37 °C. For control purposes, in addition to TRIM, slides were hybridized with H3 histone gene probes. Preparations were then washed three times 5 min with 50% formamide in 2xSSC and another three times 5 min with 1xSSC. Different hybridization stringencies were tested by using washing temperatures of 37, 42, 45 and 48 °C. Biotin was detected with fluorescein isothiocyanate (FITC) conjugated avidin and biotinylated anti-avidin (Vector) whereas digoxigenin was detected with anti-digoxigenin antibodies conjugated with tetramethylrhodamine isothiocyanate (TRITC) (Sigma). Chromosome preparations were counterstained with DAPI, mounted with antifade and examined by fluorescence microscopy. Separated images for each fluorochrome were recorded and merged as indicated above.

Single, double, and sequential FISH experiments using 5S and 28S rDNA and H3 histone gene probes were performed on metaphase chromosome spreads obtained from striped venus clams collected in all four regions. Biotin and digoxigenin labeled probes were generated either directly by PCR or by nick translation [36 (link)]. Chromosome slides were digested with RNase and pepsin before denaturation (70°C, 2 min) and hybridized overnight at 37°C. Biotin was detected with fluorescein isothiocyanate (FITC) conjugated avidin and biotinylated anti-avidin (Vector) whereas digoxigenin was detected with anti-digoxigenin antibodies conjugated with tetramethylrhodamine isothiocyanate (TRITC) (Sigma). Chromosome slides were counterstained with 4′-6-diamidino-2-phenylindole (DAPI, 0.14 μg/mL in 2xSSC) and mounted with antifade (Vectashield, Vector).
Chromosome preparations were examined with a Nikon Eclipse-800 microscope equipped with an epifluorescence system [36 (link)]. Separated images for each fluorochrome were recorded and pseudocolored using a DS-Qi1Mc CCD camera (Nikon) controlled by the NIS-Elements software (Nikon). Merging of the images was performed with Adobe Photoshop.