Anti digoxigenin rhodamine fab fragments

Larval neuroblast chromosomes from Oregon R were prepared as described previously58 (link). Chromosomes were counterstained with 4′,6-diamino-2-phenylindole (DAPI). The dodeca satellite oligo probe 5′-CCCGTACTGGTCCCGTACTGGTCCCGTACTCGGTCCCGTACTCGGT-3′ and the 10 bp satellite oligo probe 5′-AATAACATAGAATAACATAGAATAACATAGAATAACATAGAATAACATAG-3′ were chemically synthesized and labeled at the 5′ end with Cy3 or at the 3′ end with fluorescein (New England Biolabs). DNA probes derived from clones or PCR products were labeled by nick translation with digoxygenin-11-dUTP (Roche) using the DIG-Nick Translation Mix (Roche). Digoxygenin labeled probes were detected with Anti-Digoxigenin-Rhodamine, Fab fragments (Roche) in a 1:200 dilution, following supplier recommendations. Digital images were obtained using a Zeiss Axiover 200 microscope equipped with a cooled Charge-Coupled Device camera. The fluorescent signals were recorded separately as grey-scale digital images and then pseudo-colored and merged using Adobe Photoshop software.

Metaphase I (MI) chromosome spreads from root-tip cells and pollen mother cells
(PMCs) were prepared as described by Gill et al.
(1991). Total genomic DNA was extracted following the method of Yang et al. (2006 ). GISH was performed
according to the protocols described by Jiang
and Gill (1993). FISH was performed according to the method of Jiang et al. (1994 ). The
oligonucleotide probes pSc119.2 and pAs1 were labeled with biotin-16-dUTP and
digoxigenin-11-dUTP, respectively (McIntyre et
al., 1990; Nagaki et al.,
1995; Du et al., 2017 (link)).
Anti-digoxigenin-rhodamine Fab fragments (Roche Diagnostics GmbH, Germany) and
streptavidin-fluorescein thiocyanate (FITC) (Roche Diagnostics GmbH, Germany)
were used, followed by staining with 4,6-al amidine-2-phenyl indole (DAPI) to
detect digoxigenin and biotin signals, respectively. Signals were visualized
under an Olympus BX60 Fluorescence microscope (Olympus Optical Co. Ltd, Tokyo,
Japan). Images were captured with a SPOT 32 CCD camera (SPOT Charge Coupled
Device, Diagnostic Instruments, Inc., Sterling Heights, MI, USA) and analyzed
using Adobe Photoshop software.

DNA extraction was performed using PureLink Genomic DNA Mini Kit (Invitrogen) following the manufacturer’s instructions. The probes were obtained from a PCR using genomic DNA of Peckoltia sp. 3 Jarumã and Peckoltia sp. 4 Caripetuba with primers previously described for 18S rDNA (Hatanaka and Galetti Jr, 2004 (link)), for 5S rDNA (Suarez et al., 2017 (link)) and U1 snDNA (Cabral-de Melo et al., 2012 (link)). These probes were labeled by nick-translation with biotin or digoxigenin. Telomeric probes were obtained from PCR using the set of primers F-5′(TTAGGG)₅-3′ and R-5′(CCCTAA)₅-3′ followed by labeling with Digoxigenin-11-dUTP (Roche Applied Science^®) (Ijdo et al., 1991 (link)). Fluorescence in situ hybridization (FISH) was performed as described by Martins and Galetti (1999) (link) using the following stringency conditions: 2.5 ng/μL of each probe, 50% formamide, 2 x SSC, 10% dextran sulfate, and hybridization at 42°C for 16 h. Fluorescent signals were detected using Streptavidin Alexa Fluor 488 (Molecular Probes, Carlsbad, CA, United States) and anti-digoxigenin rhodamine Fab fragments (Roche Applied Science, Penzberg, Germany). Chromosomes were counterstained with 0.2 μg/ml of 4′6-diamidino-2-phenylindole (DAPI) in Vectashield mounting medium (Vector, Burlingame, CA, United States).

Polytene chromosomes were squashed from salivary glands of third instar male larvae. NotI and PstI restriction enzymes were used to extract a fragment of the ZAM pol gene from a previously published plasmid [53 (link)]. The probe was labeled with digoxigenin-11-dUTP using the Nick Translation Mix (Roche #11 745 816 910), and signals were detected with anti-digoxigenin-rhodamine Fab fragments (Roche). The fluorescent in situ hybridization method was adapted from a previously described protocol [54 (link)].

FISH analysis of single-stranded oligo probes was performed according to a previous protocol (Han et al., 2015 (link)). Biotin- and digoxigenin-labeled probes were detected using streptavidin, Alexa Fluor^TM 488 conjugate (Invitrogen) and anti-digoxigenin Rhodamine Fab fragments (Roche), respectively. Chromosomes were counter-stained with 4′−6-diamidino-2-phenylindole (DAPI) in Vectashield antifading solution (Vector Laboratories) under a cover slip. Slides were examined under a Zeiss Imager M1 microscope. Images were captured and merged using MetaSystems Isis software with a CCD camera (MetaSystems CoolCube 1) attached to a Zeiss Imager M1 microscope. After the first-round FISH, slides were immersed in 1× phosphate-buffered saline (PBS) to remove the coverslips and were dehydrated in ethanol series (70, 90, and 100%, each for 5 min). Then the repeated FISH was performed according to a previous procedure (Cheng et al., 2001 (link)).

Indirect immunofluorescence was performed on root cells or isolated nuclei from leaves as described previously (Fujimoto et al., 2004 (link); Tirichine et al., 2009 (link)). CENH3 was detected with rabbit anti-HTR12 antibody (Talbert et al., 2002 (link)) (1:1 000) and GFP was detected with either rat anti-GFP antibody (1:200 Bio Academia 1A5) or rabbit anti-GFP antibody (1:200 Invitrogen A11122). The signals were amplified using Alexa 488 anti-rat antibody (1:200, Life Technologies) and Alexa 546 anti-rabbit antibody (1:200, Life Technologies) or Alexa 488 anti-rabbit antibody (1:200, Life Technologies). Cells were counterstained with DAPI in VECTASHIELD anti-fade mounting medium (Vector Laboratories).
FISH was performed essentially as described previously by Shibata and Murata (2004) (link) following immunofluorescence staining. Briefly, probes recognizing 180bp centromeric repeats or telomeres were synthesized by nick translation using a DIG nick translation mix (Roche Diagnostics). The DIG-labelled probes were visualized using anti-digoxigenin-rhodamine Fab fragments (Roche).