Ds qi1mc ccd camera

Coimmunofluorescence was performed on 4% paraformaldehyde preparations (49) or in 3:1 ethanol:acetic acid fixed material followed by antigen recovery (50 (link)). Primary antibodies are the following: rat anti-AtZYP1-C (51 (link)), rat/guinea pig anti-AtZYP1-C (1:500) (14 (link)), rabbit anti-AtZYP1-N (1:500) (38 (link)), guinea pig/rat anti-ASY1 (1:500) (52 (link)), rabbit anti-HvHEI10 (1:200) (53 (link)), and rat anti-AtSMC3 (1:200). Secondary antibodies at 1:200 are the following: goat anti-rabbit AMCA (Jackson ImmunoResearch), goat anti-guinea pig Alexa Fluor 488 (Abcam), goat anti-rat Alexa Fluor 488 (Invitrogen), and goat anti-rabbit DyLight 594 (Vector laboratories). A Nikon Ni-E fluorescence microscope with a DS-Qi1MC CCD camera and NIS-Elements software was used to capture images. Super-resolution microscopy methodology is provided in SI Appendix.

Mitotic metaphase and meiotic prophase I chromosome preparations were obtained as it follows: after treating bivalve specimens with colchicine (0.005%) for 12 h, gills and gonads were excised, immersed in 50% and 25% seawater for 1 h and fixed in a mixture of 75% absolute ethanol and 25% acetic acid for 1 h. Small pieces of fixed material were then treated with 60% acetic acid to obtain cell suspensions that were dropped onto slides heated to 50 °C^{41 ,42} .
Bivalve synaptonemal complexes were prepared from male specimens^{41 ,43 (link)}. Meiotic cell suspensions were dropped onto glass slides, treated with 0.1 M sucrose and 0.5% Triton X-100 for 2 to 4 min, fixed with paraformaldehyde (4%) overnight, rinsed with distilled water and air-dried.
Chromosome preparations were stained with a mixture of DAPI (0.14 µg/mL) and PI (0.07 µg /mL) for 8 min, washed in tap water, air-dried and mounted with antifade (Vectashield, Vector). Slide visualization and photography were performed using a Nikon Eclipse-800 microscope equipped with an epifluorescence system. Separated images for each fluorochrome were obtained with a DS-Qi1Mc CCD camera (Nikon) controlled by the NIS-Elements software (Nikon). Merging of the images was performed with Adobe Photoshop CS2 (Adobe Systems).

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.