The preparations of mitotic metaphase and meiotic pachytene chromosomes were conducted using modified Carnoy’s solution II in accordance with the work of Setiawan et al. [13 (link)]. The Cmcent probe was labeled with Biotin-Nick translation mix (Roche), whereas CmSat162 and CmSat189 were labeled with Dig-Nick translation mix (Roche). The FISH protocol as described by Setiawan et al. [13 (link)] was followed. For the pachytene chromosomes, the hybridization mixtures were added on the chromosome preparations, covered with a 22 x 40-mm cover slip and sealed with rubber cement. The slides were denatured on a hot plate at 80°C for 2–3 min. Finally, the slides were placed in a humidity chamber and incubated at 37°C overnight. Detection solutions of 126 μL [1% BSA in 4x SSC 125 μl + 0.4 μl/ml anti-digoxigenin rhodamine (Roche) 0.5 μL + 0.5 μg/mL biotinylated streptavidin-FITC (Vector Laboratories) 0.5 μl] were used and washed in 2x and 0.1x SSC for 3 min after incubation at 37°C for 30 min. Finally, the slides were counterstained with 4,6-diamidino-2-phenylindole (DAPI) in a VectaShield antifade solution (Vector Laboratories).
Biotinylated streptavidin fitc
Manufactured by Vector Laboratories
Biotinylated streptavidin-FITC is a fluorescently labeled protein complex used in various bioassays. Streptavidin, a bacterial protein, binds strongly to biotin, a small molecule. The FITC (fluorescein isothiocyanate) fluorescent label allows visualization and detection of the bound complex.
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Lab products found in correlation
2 protocols using biotinylated streptavidin fitc
1
Chromosome Preparation and FISH Labeling
2
FISH Chromosome Analysis Protocol
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Chromosome slides were re-fixed in 1% paraformaldehyde in PBS for 10 min at room temperature. Procedures for FISH were followed as described by Kikuchi et al. (2008) . The 10 µL of hybridization cocktail containing 50% formamide, 10% dextran sulfate, 2 SSC, and 100-500 ng labeling probe was applied to the slide, covered with a coverslip, and then denatured for 3 min at 80 C on a heat block. After incubation at 37 C for one day, the slide was washed and briefly dried. Detection of the biotin-and DIG-labeled probes was performed with biotinylated streptavidin-FITC (Vector Laboratories) and anti-digoxigenin-rhodamine (Roche), respectively. The slides were counter-stained with 5 µg/mL of 4,6-diamidino-2-phenylindole (DAPI) in a VectaShield antifade mounting medium (Vector Laboratories). All FISH images were captured with an Olympus BX53 fluorescence microscope equipped with a CCD camera (CoolSNAP MYO, Photometrics). The FISH images were processed by MetaMorph, Meta imaging series version 7.8 (Molecular Devices, San Jose) and edited with Photoshop CC 2017 (Adobe).
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