Ab7064

For immunofluorescence evaluation on cancer cells, SKBR3 cells (3 × 10⁴/well) were plated in 6-well plates that were previously fitted with four cylindrical glasses at the base. After 24 h, cells were fixed with a 1:1 solution of methanol/acetone for 10 min at a temperature of −80 °C and then incubated with 1% bovine serum albumin in DPBS for 1 h. Cells were firstly incubated with the primary antibody Actin, (1:50) for 2 h at room temperature (Sigma-Aldrich Cat# A2228), and afterwards with a FITC labeled secondary mouse antibody for 1 h at room temperature (Abcam Cat# ab7064). The KCTD15 primary antibody (1:50) (GeneTex Cat# GTX50002) was incubated simultaneously. A PE-labelled secondary rabbit antibody (Sigma-Aldrich Cat# P9537) was used for the detection of KCTD15. Nuclei were stained in blue with DAPI (1:30,000) for 10 min at room temperature (Thermo Fisher Scientific Cat# D1306). For immunofluorescence and phase-contrast images, cells were pictured using an automated upright microscope system (Leica DM5500 B) coupled with Leica Cytovision software.

Nuclear immunofluorescence experiments for AtMORC4/7-MYC tagged lines were performed based on the method described in [25 (link)]. Leaves from three-week old plants were fixed in 4% paraformaldehyde in TRIS buffer (10 mM TRIS pH 7.5, 10 mM EDTA, and 100 mM NaCl) for 20 minutes and washed twice in TRIS buffer. Leaves were chopped in 200–400 microliters lysis buffer (15 mM TRIS pH 7.5, 2 mM EDTA, 0.5 mM spermine, 80 mM KCl, 20 mM NaCl, and 0.1% Triton X-100) and filtered through a 3 μM cell strainer (Corning, #352235). 5 μL of nuclei suspension was added to 12 μL of sorting buffer (100mM TRIS pH 7.5, 50mM KCl, 2mM MgCl2, 0.05% Tween-20, and 20.5% sucrose) and air dried on chloroform dipped microscope slides for two hours and then post-fixed in 4% paraformaldehyde in PBS for 20 minutes. Slides were washed three times in PBS and incubated in blocking buffer (3% BSA, and 10% horse serum in PBS) for 30 minutes at 37°C. Nuclei were incubated at 4°C overnight in mouse monoclonal antibody against c-Myc (9E10, Abcam ab32; 1:200). Slides were washed in PBS and incubated with goat anti-mouse FITC antibody (Abcam, ab7064; 1:200) for 90 minutes at room temperature. Following PBS washes, nuclei were counterstained and mounted in Vectashield mounting media with DAPI (Vector, H-1200). Nuclei were analyzed with a Zeiss LSM 710 Confocal microscope at 63X or 100X magnification using Zen software.

BM smears of B-ALL patients were air-dried overnight and then fixed at −80 °C for 15 minutes with a −80 °C pre-cooled solution of Methanol-Acetone 1:1 (v/v). Subsequently, the slides were subjected to blocking with solution of 3% (w/v) BSA in PBS pH 7.4 at Room Temperature (RT). Anti KCTD15 monoclonal antibody was diluted 1:100 in a solution of PBS + 1% (w/v) BSA and then all slides were incubated for 4 h at + 4 °C. After three wash steps in PBS for 5 minutes each, FITC- conjugated anti-mouse secondary antibody (ab7064, Abcam, UK) diluted 1:200 in a solution of PBS + 1% (w/v) BSA was incubated for 1 h at 4 °C in the dark. After additional three wash steps in PBS, a solution of 4′,6-Diamidino-2-Phenylindole, Dihydrochloride (DAPI, Thermo Fischer Scientific D1306) diluted 1:35000 in PBS was used for coloring of the nuclei.
Images were obtained using an automated upright microscope system (Leica DM5500 B) coupled with Leica Cytovision software.