Concomitant fluorescence in situ hybridization (FISH) and immunofluorescenceParaffin sections were deparaffinized in a xylene series followed by heat mediated antigen retrieval. Slides were then dehydrated in an ethanol series (70%, 80% and 100%) and air dried. Frozen sections were fixed in ice-cold pure ethanol for 10 min and air-dried prior to in situ hybridization. Purified human islets were cultured on rat tail type I collagen coated coverslips. They were fixed in 1% paraformaldehyde for 15 min, then simultaneously permeabilized and blocked in 0.1% (w/v) saponin (Sigma, S4521), 3% BSA solution for 45 min, and subsequently dehydrated in ethanol series. After pre-treatments, tissue sections or cell preparations were incubated with X/Y chromosome FISH probes (Vysis CEP X Spectrum Orange™/Y Spectrum Green™ Direct labelled Fluorescent DNA probe, Abbot Molecular Inc., Des Plaines, IL) as described previously [10]. To study aneuploidy/polyploidy, the CEP 18 probe (Abbot Molecular Inc.) was used in combination with the X/Y probe. After post-hybridization washes, sections or cells were incubated with primary antibodies (and controls as appropriate) specific for insulin (DAKO, A0564), glucagon (DAKO, A0565), somatostatin (Santa Cruz, SC-13099), GATA4 (Santa Cruz, SC-1237), Cytokeratin-19 (Abcam, Ab9221), Ki67 (Abcam, Ab833), CD68 (DAKO, m0876), CD45 (DAKO, M0701), nestin (Millipore MAB5326), or CD34 (Invitrogen, 073403) for 2 hrs at 37°C. After probing with the corresponding fluorescent secondary antibody for 1 hr at room temperature, sections were washed three times in 1× PBS solution, dehydrated, and counterstained with DAPI permanent mounting media (VECTASHIELD®, Vector laboratories, CA).Image analysisFluorescent imaging Images were captured using an Olympus BX41 fluorescent microscope fitted with an AxioCam MRm digital camera (Zeiss, Germany). The X and Y chromosomes were analyzed using the ISIS fluorescent imaging software (Zeiss, Germany) designed for FISH.Confocal microscopy To confirm MMc frequencies, pancreatic tissue sections were screened using a Leica SP5 confocal imaging system at the Wolfson Bioimaging Facility, School of Biochemistry, University of Bristol, UK. Z-stack images of each tissue section were analyzed for FISH and immunofluorescence to allow visualization of X and Y chromosome signals in all planes of the nucleus.Counting strategyFISH signals were checked in individual filter channels to ensure signal fidelity and that the visualization of two red dots representing two copies of the X chromosome was not caused by immunofluorescence cross talk. Only cells that showed clear XY (red and green dot) or XX (two red dots) signals within single nuclei were counted. Any cells with overlapping nuclei were excluded from the analysis. X chromosome “splits” that occasionally appeared as two juxtapositional dots due to DNA breakage were not considered as XX signals. A FISH success rate (the frequency of nuclei with two clear signals visible) of greater than 60% was required before further analysis.All T1D and matched control tissues were stained for FISH and insulin. Overall, as many nuclei as possible were counted (>1000 at least), including at least 20 islets per section. MMc were counted in insulin positive and insulin negative cell fractions. For T1D case 1, 2, and 3, MMc were also examined in CD45+ population. An additional independent, blinded scorer reviewed each candidate female cell.