Vectashild

The physical localization of the 6 tandem repeats (TRs) was analyzed by the FISH procedure. A chromosome spread preparation was made from root tip as described in [28 (link)]. Good slides with well-spread chromosomes were stored at 4 °C until use. FISH was carried out following the procedure in [60 (link),61 (link)]. After hybridization, the chromosomes were counterstained with 1 mg/mL DAPI. The detection was performed using streptavidin-conjugated Cy3 or FITC (Roche, Basel, Switzerland); signals in all variants were visualized using an AxioZeiss Imager V1 fluorescence microscope.
After post-hybridization washing of WWGH chromosome mitotic preparations as described in Komuro et al. [62 (link)] multicolor genomic in situ hybridization (mcGISH) was conducted on the same slides as described in Kishii et al. [63 (link)] with modifications described in Salina et al. [15 (link)]. The probes were the Pseudoroegneria spicata (St genome) and Dasypyrum villosum (V genome) genomic DNA (50 ng/preparation) labeled with digoxigenin-11-dUTP and with biotin-16-dUTP, respectively, by nick translation, according to the manufacturer’s instructions (Roche, Germany). The chromosomes were counterstained with 1 mg/mL DAPI and mounted in Vectashild (Vector Laboratories, Peterborough, UK).
The results were recorded with an AxioCam Mrm Zeiss camera and contrasted using AxioVision software.

Tissue samples were immersed in 4% formaldehyde overnight. Subsequently, they were washed three times with 1X phosphate-buffered saline (PBS), and then cryopreserved in 20% sucrose for 3 days and 40% sucrose for 1 week. Samples were then embedded in OCT (Merck KGaA, Darmstadt, Germany) and sectioned using a Leica CM1860 cryostat (Leica Biosystems, Leider Lane Buffalo Grove, USA). The sections were then subjected to immunofluorescence. Primary antibodies specific for keratinocyte markers were used: cytokeratin 10 (abcam, Cambridge, MA, USA, ab76318), cytokeratin 6 (abcam, ab93279), and EGFR (Santa Cruz, Santa Cruz Biotechnology, Texas, USA, sc-373746). The sections were incubated with primary antibodies, diluted 1/50, overnight, after which they were washed with PBS 7.4 and then incubated with secondary antibodies, diluted 1/100, for 2 hours (protected from light). The slides were assembled using vectashild® (Vector Laboratories, CA, USA) and coverslips. The analyzes were performed with confocal microscopy (Leica Microsystems, Teban Gardens Crescent, Singapore).

HMEC cultured on collagen-coated 1.5 coverslips for 24 hours were washed with 3X PBS, fixed in 4% paraformaldehyde for 10 minutes, permeabilized in 0.1% Triton for 10 minutes, and blocked in 1% BSA/1X PBS for 30 minutes. After washing cells in 3X PBS, they were serially incubated in Anti-Pyruvate Kinase antibody (1:500, Abcam, MA, US, ab6191), Goat antibodies were conjugated to a secondary Alexa 488 antibody 1:500. The coverslips were then mounted on microscope slides using Vectashild (Vector Laboratories, Burlingame, CA, USA) containing 4',6-diamidino-2-phenylindole (DAPI) for nuclei staining. Images were captured with a Leica TCS SP5 (Leica Microsystems Germany) confocal microscope.