Anti digoxigenin rhodamine

FISH experiments were performed with eight satDNA families, which were common to the four analyzed species. We utilized primers described by Silva et al. (2017) (link) and probes were labeled with digoxigenin-11dUTP in PCR reactions. FISH experiments were performed following the protocol established by Pinkel et al. (1986) (link), with some modifications (Utsunomia et al., 2017 (link)). The metaphasic plate was treated with RNase A (50 μg/ml), for 50 min, with subsequent chromosomal DNA denaturation in 70% formamide/2 × SSC for 2 min, at 70°C. After hybridization, the slides were washed in 0.2 × SSC/15% formamide for 5 min at 42°C, with subsequent washes in 4 × SSC/0.5% Tween-20, at room temperature. Probe detection was performed with anti-digoxigenin-rhodamine (Roche, Basiléia, Switzerland) and the chromosomes were counterstained with DAPI (4ʹ,6-diamino-2-phenylindole, Vector Laboratories, Burlingame, United States). The results were analyzed using an optical microscope (Olympus BX61). Images were captured using the DP Controller software (Olympus®, Hamburg, Germany).

DNA FISH probes for Ret and Gfra1 were labeled with digoxigenin by a Nick Translation kit (Roche) according to the manufacturer’s protocol using bacterial artificial chromosome DNA clones: Ret (RP23-98B12; Thermo Fisher Scientific) and Gfra1 (RP23-180P13; Thermo Fisher Scientific). Whole-chromosome painting probes for mouse Chr. 6 and Chr. 10 were purchased from MetaSystems Probes. For DNA FISH, cells were grown on coverslips, fixed with 4% formaldehyde for 10 min, and permeabilized with 0.5% Triton X-100 for 10 min. Further, cells were incubated with 20% glycerol/PBS for 60 min followed by treatment with 0.1 N HCl for 20 min and washes with 2× saline-sodium citrate (SSC) buffer (1× SSC: 0.15 M NaCl and 0.015 M sodium citrate, pH 7.0) for 5 min and 50% formamide/2× SSC for 30 min. Next, cells were hybridized with a labeled DNA probe for 24 h at 37°C. Probes were detected by anti–digoxigenin-rhodamine, Fab fragments (Roche), and cell nuclei were counterstained with DAPI. Images were acquired using LSM 700 (Carl Zeiss Micro Imaging) with a 63× objective and analyzed with ImageJ (National Institutes of Health) or Zeiss ZEN Microscope Software (Carl Zeiss Micro Imaging).

FISH with the 5S and 45S rDNA probes, and cGISH with V.umbellata genomic DNA probe were performed after CPD staining on the same slides. The slides previously stained by CPD were washed in 2× SSC, twice for 15 min each, dehydrated through an ethanol series (70%, 90%, and 100%, 5 min each) and then used for hybridization. The in situ hybridization methodology followed the protocol described by She et al. (2015) (link). The biotin-labelled probe was detected using Fluorescein Avidin D (Vector Laboratories, Burlingame, USA). The digoxigenin-labeled probe was detected by anti-digoxigenin-rhodamine (Roche Diagnostics, Mannheim, Germany). The preparations were counterstained and mounted with 3 µg ml⁻¹DAPI in 30% (v/v) Vectashield H-1000 and examined under the epifluorescence microscope mentioned above. Grey-scale images were digitally captured using METAMORPH software with UV, blue and green filters for DAPI, fluorescein, and rhodamine, respectively. The images were then merged and edited with ADOBE PHOTOSHOP version 8.01.

Zebrafish embryos were euthanized, fixed, permeabilized and re-fixed as described for whole-mount immunofluorescence. For the terminal deoxynucleotidyl transferase (TdT) dUTP nick-end labeling (TUNEL) assay, the ApopTag^® Red In Situ Apoptosis Detection Kit (Millipore Sigma, #S7165) was used. Briefly, fixed and permeabilized embryos were incubated in equilibration buffer for 1 h at room temperature, and then incubated in a reaction mix (20 ml of equilibration buffer, 12 ml of reaction buffer, 6 ml of TdT enzyme, 0.5 ml of 10% Triton X-100) overnight at 37°C. Larvae were then washed several times with PBST and then incubated in blocking solution (10 mg/ml BSA, 2% BFS, 1% DMSO, 0.1% Triton X-100). Anti-digoxigenin-rhodamine (1:50, Roche, #11207750910) was used as conjugated primary antibody. Quantification was performed as described above.

FISH and sperm-FISH procedures were carried out as described in Vozdova et al. 2019 [32 (link)]. BAC probes labeled with digoxigenin-11-dUTP were detected with antidigoxigenin rhodamine (Roche Diagnostics, Indianapolis, IN, USA). BAC probes labeled with biotin-16-dUTP were detected with Avidin-FITC (Vector Laboratories, Inc., Burlingame, CA, USA). Hybridization signals were examined using Zeiss Axio Imager.Z2 fluorescence microscope (Carl Zeiss Microimaging GmbH, Jena Germany) equipped with appropriate fluorescent filters and the Metafer Slide Scanning System (MetaSystems, Altlussheim, Germany). Images of well-spread metaphase cells were captured and analyzed using ISIS3 software (MetaSystems, Altlussheim, Germany).

Meiotic cells were obtained from testes, whereas mitotic cells were obtained from embryos. For conventional analyses used to check the general chromosomal structure of animals from different populations, the slides were prepared with tissue maceration and staining with Giemsa 5%.
The PCR products from satDNAs from Rio Claro/SP individuals were used as probes for FISH assays using chromosomes of individuals from the populations of Rio Claro/SP, Cabo/PE, Sta Bárbara do Pará/PA and Posadas/AR. Fragments were labeled by nick translation using digoxigenin-11-dUTP and detected by anti-digoxigenin rhodamine (Roche, Mannheim, Germany) or biotin-14-dUTP detected with streptavidin Alexa Fluor-488 conjugated (Invitrogen, San Diego, CA, USA). FISH experiments were conducted following [77 (link)], with adaptations by [78 (link)]. Fiber-FISH was conducted according to [79 (link)]. Slides were counterstained with DAPI (4′,6-Diamidine-2′-phenylindole) and mounted with VECTASHIELD (Vector, Burlingame, CA, USA). Pictures were captured using a DP70 cooled digital camera in gray scale coupled with an Olympus microscope BX51 equipped with a fluorescence lamp and appropriate filters. The images were pseudo-colored, merged and treated for brightness and contrast using Adobe Photoshop CS6.