Telomeric Immuno-FISH Protocol for Meiotic Proteins

Immuno-FISH with telomeric probe was performed sequentially to the immunodetection of meiotic proteins, according to the protocol described [55 (link)]. Denaturation of chromosomal DNA and probes occurred at 70 °C and 100 °C, respectively. Slides were kept at 37 °C, overnight, for hybridization. Subsequently, slides were washed with 2xSSC and 4xSSC-Tween to remove nonspecific hybridizations. Probes were detected with anti-digoxigenin-FITC. Chromosomes were counterstained with 4-6-diamidino-2-phenylindol (DAPI) in VECTASHIELD antifading solution (Vector). Images were captured using an AxioCam MRm CCD camera (Nikon, Tokyo, Japan) attached to a Nikon H550S epifluorescence microscope (Nis-Elements AR program (Nikon, Tokyo, Japan)) and the Nis-Elements software; all images were photographed using 100× magnification objective lenses.