To confirm the fetal origin of the CV-MSC isolated from placentas collected after the birth of male newborns, X/Y chromosome analysis was performed at early (p3–p4) and late (p8–p10) passages for all donors used for different subsequent experiments (n = 5) (Fig. 1a iv–v). The ZytoLight CEN X/Y Dual Color Probe (Zytomed Systems, Germany) was used for detection of human α-satellites of X and Y chromosomes by fluorescence in-situ hybridization (FISH). Ten microliters of the hybridization mixture was added to the cytospun cells, and DNA denaturation was then performed at 75 °C for 2 minutes with subsequent overnight incubation using a humidified chamber. After hybridization, the cytospins were washed with cytology stringency buffer for 2 minutes at 72 °C and subsequently rinsed in sodium chloride/sodium citrate buffer for 1 minute at room temperature (RT). Slides were finalized using Vectashield antifade mounting medium containing 4′,6-diamidino-2-phenylindole (DAPI) (Vector Labs, Germany) and fluorescence detected on an Axiovert S135 microscope (Zeiss, Germany). The α-satellite sequences of the centromere of chromosome X were excited at 488 nm and of chromosome Y at 554 nm.
Axiovert s135 microscope
Manufactured by Zeiss
Sourced in Germany
The Axiovert S135 is an inverted microscope designed for routine observation and documentation. It features a stable stand, a binocular observation tube, and a mechanical stage for sample positioning. The microscope is equipped with objectives for bright-field microscopy, allowing users to view and capture images of specimens.
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2 protocols using axiovert s135 microscope
1
Fetal Origin Verification of CV-MSCs
2
FISH-based Detection of MDM2 Amplification
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To validate NGS-based detection of MDM2 gene amplification, fluorescence in situ hybridization was performed on corresponding formalin-fixed, paraffin-embedded tissue samples using the ZytoLight SPEC MDM2/CEN 12 Dual Color Probe (ZytoVision GmbH, Bremerhaven, Germany). The test is based on the use of two fluorescently labeled oligonucleotide probes: a MDM2-specific probe (green fluorochrome ZyGreen) and a probe targeting the centromeric region of chromosome 12 (orange fluorochrome ZyOrange) on which the MDM2 locus is located. The test was performed according to the manufacturer's instructions. In brief, deparaffinized and rehydrated tissue sections were immersed in pretreatment buffer for 20 minutes at 98 C, followed by enzymatic pepsin digestion for 10 minutes at 37 C. Subsequently, the dual-color probe was applied to the tissue sections, followed by DNA denaturation at 75 C and a hybridization step at 37 C overnight. After stringent washing and DAPI counterstaining, hybridized probes were visualized by fluorescence microscopy (Axiovert S135 microscope; Zeiss). A minimum of 50 nuclei per case was analyzed, and the mean ratio of green (MDM2) over orange (CEN 12) hybridization signals was calculated. A case showing a ratio of >2.0 was considered MDM2-amplified.
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