Dmi6000 cs

Whole-mount FISH was performed on gonads dissected from tadpoles according to Dedukh et al.⁴⁸ . Gonadal tissues were incubated in 0.5% solution of Triton X100 in 1× PBS for 4–5 h, washed in 1× PBS and impregnated in 50% formamide, 10% dextran sulfate, and 2× SSC for 3–4 h at 37 °C. Hybridization was performed either only with probe to RrS1 (20 ng/µl) or with the usage of the mixture of probes to RrS1 and PlesSat01-48 repeats (10 ng/µl of each probe). Hybridization mixture contained 50% formamide, 2× SSC and 10% dextran sulfate and salmon sperm DNA (with 10–50-fold excess compared to the probe concentration). After denaturation of tissues at 72 °C for 15 min and their incubation for at least 24 h at RT, tissues were washed three times in 0.2× SSC at 50 °C for 15 min each. Afterwards tissues were transferred in 1% blocking solution (Roche) prepared in 4× SSC for 1 h at RT. Biotin and digoxigenin were detected by streptavidin conjugated with Alexa 488 (Invitrogen, San Diego, CA, USA) and anti-digoxigenin antibodies conjugated with rhodamine (Invitrogen, San Diego, CA, USA), correspondingly. After incubation with streptavidin and rhodamine overnight at RT, tissues were washed in 4× SSC for 15 min tissues and stained with DAPI (1 µg/µl) (Sigma) prepared in 1× PBS at RT overnight. Prior examination, tissues were placed in a drop of Vectashield antifade solution and analyzed using Leica TCS SP5 confocal laser scanning microscope based on the inverted microscope Leica DMI 6000 CS (Leica Microsystems, Germany). Diode, argon and helium-neon lasers were used to excite the fluorescent dyes DAPI, fluorochromes Alexa488 and rhodamine, respectively. LAS AF software (Leica Microsystems, Germany) was used for image capture and processing. Based on the FISH signals, spermatids with two L signals and weak R signals (based on the exposition value) were evaluated as haploid L spermatids; spermatids with no L and strong R signals were assessed as haploid R spermatids; and spermatids with one and three signals of L probe together with strong or weak signals of R probe were assessed as aneuploid. Metaphases with a combination of R and L univalents and metaphase plates exhibiting abnormal pairing of R and L chromosomes in bivalents were classified as unusual. Metaphase plates with incomplete chromosome numbers (less than 13 in haploid stages or less than 26 in diploid stages) were identified as aneuploid. Based on previous studies of various water frog populations^{31 (link),48} , we conservatively estimate a rate of 15% for “mistakes” in micronuclei with incorrectly eliminated genomes. In these populations, cells with incorrectly eliminated chromosomes did not produce fertile gametes and were not represented in the progeny. In our case, we use a threshold of monospermic males as of those with observed up to 15% of R : L spermatids.

One × 10⁴ cells were seeded in 12-well cell culture plates and transfected with each siRNA as described. When needed, samples were also transfected after 24 h, using Lipofectamine LTX (Life Technologies). Next, cells were washed with PBS, fixed with ice-cold methanol for 10 min, permeabilized with 0.2% Triton X-100 solution for 20 min and then blocked for 20 min with 2.5% BSA in PBS. Cells were then incubated with appropriate primary antibodies for 30 min, washed three times with PBS, and then incubated with appropriate Alexa Fluor 488-conjugated (Invitrogen, A21202) or Alexa Fluor 555-conjugated (Invitrogen, A31570) secondary antibodies. Nuclei were stained with 1.5 μM 4′,6-diamidino-2-phenylindole (DAPI) in PBS for 5 min. Coverslips were mounted in fluorescence mounting medium (Dako, S3023). Samples were visualized on a TSC SP5 confocal microscope (Leica, 5100000750) installed on an inverted LEICA DMI 6000CS (10741320) microscope using an oil immersion PlanApo 40 × 1.25 NA. Images were acquired using the LAS AF acquisition software (Leica).