Cy3 labeled streptavidin

The plasmid
that codes biotin-labeled kinesin 401 (K401) was a gift from Jeff
Gelles (pWC2 – Addgene plasmid #15960; http://n2t.net/addgene: 15960;
RRID_Addgene_15960)^{21 (link)} and was purified
according to previously published protocols.^{22 (link),23 (link)} To create active kinesin clusters, multimotor complexes were prepared
by mixing 0.2 mg/mL kinesin-1 and 0.1 mg/mL streptavidin (Sigma, S4762)
in M2B buffer (M2B: 80 mM PIPES, adjusted to pH = 6.8 using KOH, 1
mM EGTA, 2 mM MgCl₂) containing 0.9 mM DTT and incubated
on ice for 15 min. Four microliters of this mixture were combined
with 1% PEG and 2 mM ATP. The final concentration of kinesin in the
solution was 17 nM. To maintain a steady ATP concentration for the
entire experimental duration, an ATP regeneration system containing
32 mM phosphoenol pyruvate (PEP, Alfa Aesar B20358) and 1.7 μL
of pyruvate kinase/lactic dehydrogenase enzymes (PK/LDH, Sigma, P-0294)
was incorporated. To reduce photobleaching effects, an oxygen scavenging
mix containing 0.2 mg/mL glucose oxidase (Sigma, G2133), 0.05 mg/mL
catalase (Sigma, C40), 2.4 mM Trolox (Sigma, 238813), 0.5 mg/mL glucose,
and 0.65 mM DTT was included. For experiments with labeled motor clusters,
0.1 mg/mL Cy-3-labeled streptavidin (Sigma, S6402) was used to create
the kinesin complexes.

Young ovaries for chromosome staining and counting and inflorescences for meiotic study were collected and fixed in a mixture of ethanol: acetic acid (3:1) for 24 h and then transferred to 70% ethanol and refrigerated until use. The determination of chromosome numbers and meiotic observations were made according to the method of Li et al. (1995) [35 (link)].
Slide preparations of chromosomes for GISH mainly followed the procedures of Zhong et al. (1996) [36 (link)] and Ge et al. (2007) [37 (link)]. In situ hybridization was carried out according to the protocols of Leitch et al. (1994) [38 ]. Hybridization signals of the O. violaceus probe were detected using Cy3-labeled streptavidin (Sigma, St. Louis, MO, USA) and chromosomes were counterstained with 0.2% 4′-6-diamidino-2-phenylindole (DAPI) solution (Roche, Basel, Switzerland), mounted in antifade solution (Vectashield), and examined under a Zeiss fluorescent microscope (Axio Scope A1, Munich, Germany) equipped with a CCD camera. Images were processed using Adobe Photoshop (8.0) to adjust contrast and brightness.