4 6 diamidino 2 phenylindole (dapi)

After meiotic spread preparation and FISH procedures, chromosome slides were counterstained with 0.1 μg/mL DAPI (4′,6-diamidino-2-phenylindole) (Serva, Heidelberg, Germany) dissolved in Vectashield mounting medium (Vector laboratories, Peterborough, UK).

IMCD3 cells stably expressing GFP-tagged protein were plated on poly-L-lysine coated coverslips in six-well plates, each well containing 100,000 cells. Twenty-four hours later, cilia were induced by 48 h serum starvation. Cells were washed in PBS and fixed with 4% formaldehyde in cytoskeletal buffer (2,75 M NaCl, 100 mM KCl, 25 mM Na₂HPO₄, 8 mM KH₂PO₄, 40 mM MgCl₂, 40 mM EGTA, 100 mM PIPES, 100 mM Glucose, pH 6.0) for 20 min. After two washes with PBS cells were permeabilized with 0.3% Triton X100 in cytoskeletal buffer for 10 min. Cells were rinsed in 0.1% Tween20 in PBS and blocked in 10% FBS in PBS for 30 min. For immunostaining of primary cilia, mouse 6-11B-1 anti-acetylated tubulin antibody (1:5,000; Sigma T6793) in 10% FBS in PBS was incubated overnight at 4 °C. Alexa Fluor 647 anti-mouse secondary antibody (1:800; Life technologies A-31571) was added for 45 min at room temperature after washing four times with 0.1% Tween20 in PBS. Coverslips were rinsed three times in 0.1% Tween20 in PBS and afterwards in PBS. Nuclei were stained with DAPI (Serva), diluted 1:10,000 in PBS for 1 min. After three washes with PBS, coverslips were fixed on glass slides with Mowiol (Merck). Images were taken using an Olympus IX81 microscope with a CCD camera and a 60x NA 1.35 oil immersion objective.

After fixation, discs were washed in 500 μL 0.9% NaCl + RNase Inhibitor and dabbed off on a paper towel. Pre-treatment of Gram-positive bacteria occurred as described before [23 (link)] within 1 mg/mL lysozyme solution in 0.1 M Tris-HCl, pH 7.5, 5 mM EDTA for 8 min at room temperature (RT). To permeabilize the cell walls accordingly Staphylococcus aureus cells needed a longer and stronger pre-treatment with 10 mg/mL lysozyme for 50 min at 37 °C and additionally with 20 µg/mL lysostaphin for 5 min at RT both in the same buffer as described previously. Pre-hybridization in 500 μL of proportionate hybridization buffer (Table 2) for 15 min at 46 °C. Immediately thereafter, the discs were transferred in extra wells with 370 μL preheated appropriate probes in the corresponding hybridization buffer (Table 2). The discs were hybridized for 4 h at 46 °C, then immersed in 2 mL preheated washing buffer and incubated for 45 min at 48 °C. Total DNA was stained with 15 µM Syto 59 (Thermo Fisher Scientific, Waltham, Massachusetts, USA) in nanopure water for 30 min or with 0.5 µg/mL DAPI (SERVA Electrophoresis GmbH, Heidelberg, Germany) in nanopure water for 5 min at room temperature. All incubations with fluorescent dyes were performed in the dark. Discs were embedded upside down on chamber slides in a matching drop of Mowiol and stored for at least 24 h before microscopic examination.

Expression plasmids expressing human MEF2C and UBE3A and respective negative control plasmids were obtained from Sino Biologicals (MEF2C-HA: HG12320-CY, UBE3A-Myc:HG11130-CM, pCMV-Myc:CV014 and pCMV-HA:CV013) and used for transient transfection. Transfected HeLa cells were grown on poly-lysine coated coverslips, fixated with 4% paraformaldehyde in PBS for 10 minutes and stained with anti-Myc (M4439, Sigma-Aldrich) and anti-HA (H6908, Sigma-Aldrich) and with Alexa Fluor™ 488 goat anti–mouse and Alexa Fluor™ 488 donkey anti–rabbit antibodies (A11001 and A10040, Thermo Fisher). Nuclei were counterstained with DAPI (Serva). Images were taken with a Zeiss Axio Imager Z2 Apotome microscope with a 63x objective and analyzed in ImageJ.

The following DNA sequences were used as probes.
The details of probe labelling and the following fluorescence in situ hybridization (FISH) procedure were as described by Jenkins and Hasterok (2007) (link) with minor modifications. Centromeric and telomeric probes were mixed together, precipitated, and dissolved in a hybridization mixture containing 50% deionized formamide and 10% dextran sulphate in 2× saline sodium citrate (SSC). After denaturation (10min, 75 °C), the hybridization mixture was applied to slides with isolated nuclei and denatured again at 75 °C for 4.5min. Hybridization was performed in a humid chamber at 37 °C for about 40h. After hybridization, the slides were washed in 10% formamide in 2× SSC (2×4min, 42 °C), which is equivalent to a 79% stringency. Immunodetection of the probes that had been labelled with digoxigenin-11-dUTP was performed according to the standard protocols using fluorescence isothiocyanate (FITC)-conjugated anti-digoxigenin antibodies (Roche). The slides were mounted in Vectashield (Vector Laboratories) containing 2.5 μg ml^–1 DAPI (Serva).

After the FISH procedure, chromosome slides were stained with 0.1 μg/mL DAPI (4′,6-diamidino-2-phenylindole) (Serva, Heidelberg, Germany) in Vectashield mounting medium (Vector laboratories, Peterborough, UK). DAPI-banding analysis was used as an additional parameter for the identification of individual chromosomes [46 (link), 47 (link)].

Before FISH procedure, chromosome slides were pretreated with 1 mg/ml of RNase A (Roche Diagnostics, Mannheim, Germany) in 2xSSC at 37°C for 1 h and then washed three times for 10 min in 2xSSC. The slides were dehydrated in a series of 70%, 85%, and 96% ethanol solutions and then air-dried. The hybridisation mixture (22 μl) containing 40 ng of each labelled probe was added to each slide. Coverslips were placed on the slides and sealed with rubber cement. Slides with DNA probes were codenatured at 74°С for 5 min, placed in a moisture chamber, and hybridised overnight at 37°C. After removing the coverslips, the slides were washed with 0.1xSSC at 44°C for 8 min and with 2xSSC at 44°C for 8 min with the final 5 min wash in 2xSSC at room temperature. For oligo-(GAA)₉ probe labelled with fluorescein-12-dUTP, fluorescent signal amplification using FITC-Alexa 488 antibodies (VectorLabs, Youngstown, USA) was performed. After incubation for 60 min at 37°C with the detection mixture, the slides were washed two times with 2xSSC for 5 min and once in 1xPBS for 5 min each at room temperature. The slides were dehydrated and air-dried in the dark. After FISH procedure, the slides were stained with 0.125 μg/ml DAPI (Serva, Heidelberg, Germany) dissolved in Citifluor anti-fade solution (UKC Chem. Lab., Canterbury, UK).