Vectashield mounting medium h 1000

The cells were grown on coverslips for 24 h, washed in phosphate-buffered saline (PBS) and fixed in 4% formaldehyde in PBS for 20 min at room temperature. After washing in PBS, the cells were permeabilized in 0.1% Triton X-100 in PBS for 5 min. To block the nonspecific binding of the antibodies, cells were incubated with 3% bovine serum albumin (BSA) in PBS for 20 min. The cells were incubated with diluted antibody in 3% BSA in PBS for 1 h at room temperature. After 3 x 5 min washing with PBS, the cells were incubated with secondary antibody in 3% BSA in PBS for 1 h at room temperature in the dark. After 3 x 5 min wash with PBS, the cell nuclei were stained with 1 μg/ml Hoechst for 5 min. The coverslips were mounted with Vectashield H-1000 mounting medium (Vector Laboratories, USA) and stored in the dark at 4°C. Primary and secondary antibodies used: ΔNp63-1.1 (ΔNp63, Moravian Biotechnology, Czech Republic), SFI-6 (p63, DCS-Innovative Diagnostik-Systeme, Germany), MOC-31 (EpCAM, Abcam, UK), and goat anti-mouse IgG DyLight-488 conjugate (Thermo Fisher Scientific, USA).

Multi-color FISH was performed as previously described in (35 (link)) with minor modifications. Tissues were serially rehydrated, and the pituitary was detached from the brain. Afterwards, whole pituitaries were hybridized with the probes (0.11 – 3.17 ng/µl) for 18 hours at 55°C, and incubated with different combinations of anti-DNP- (Perkin Elmer), anti-FITC-, and anti-DIG-conjugated antibodies (Roche Diagnostics), followed by TAMRA- (Thermofisher), Cy5- (Perkin Elmer) and FITC-conjugated tyramides (Sigma). The nuclei were stained with DAPI (1:1000, 4’, 6-diamidino-2-phenylindole dihydrochloride; Sigma). The absence of labeling when using sense probes was used to confirm the specificity of the anti-sense probes. Whole pituitaries were mounted using Vectashield H-1000 Mounting Medium (Vector, Eurobio/Abcys) between microscope slides and cover slips (Menzel Glässer, VWR) with spacers (Reinforcement rings, Herma) in between for the juveniles, and between two cover slips with spacers for adults.

Immunofluorescence labeling of chromosomes assembled in extracts was performed as described previously (Kinoshita et al., 2022 (link)) with some modifications. Fixed chromosome samples were spun down onto coverslips, washed with TBS-Tx (25 mM Tris [pH 7.5], 0.15 M NaCl, and 0.1% Triton X-100) three times, and blocked with 1% BSA in TBS-Tx at room temperature for 30–60 min. For immunolabeling, coverslips were incubated with 1% BSA in TBS-Tx containing 3 µg/mL anti-XCAP-H2 (AfR202), anti-hCAP-H2 (AfR205; Ono et al., 2003 (link)), or biotin-labeled anti-hCAP-H2 at room temperature for 60 min. The coverslips were then washed with TBS-Tx three times and incubated with 1% BSA in TBS-Tx containing a secondary antibody (Alex Fluor 568-conjugated anti-rabbit IgG [1:500; ThermoFisher] or Alexa Fluor 488-conjugated streptavidin [1:2000; ThermoFisher]) at room temperature for 60 min. Finally, the coverslips were incubated with 2 µg/mL DAPI in TBS-Tx for 5 min, washed with TBS-Tx three times, and mounted onto microscope slides with Vectashield H-1000 mounting medium (Vector Laboratories). Immunofluorescence labeling of recombinant condensin I complex was processed as above except with 1 µg/mL rabbit anti-mSMC4 antibody (Lee et al., 2011 (link)) and Alexa Fluor 568-conjugated anti-rabbit IgG (1:500; ThermoFisher) as previously described (Kinoshita et al., 2022 (link)).