RNA FISH Visualization of Nuclear Protein Localization

RNA FISH was performed with digoxigenin-labeled probes as described [36 (link),53 (link),77 (link)]. BAC DNA probes used for this study were: Scml2, RP24-204O18 (CHORI BACPAC library) and Zfx, bMQ-372M23 (Mouse bMQ BAC library). BAC-containing bacteria were grown in LB-chloramphenicol culture overnight at 37°C. A standard miniprep method was used to isolate BAC DNA. Approximately 2 μg of BAC DNA was labelled using DIG-Nick Translation Mix (Roche) and precipitated with Cot-1 DNA (Invitrogen) and salmon sperm DNA (Stratagene). Frozen testis cells were permeabilized with CSK buffer (100 mM NaCl, 300 mM sucrose, 3 mM MgCl₂, 10 mM PIPES, 0.5% Triton X-100, 2 mM vanadyl ribonucleoside (New England Biolabs)), fixed with 4% paraformaldehyde, and dehydrated through an ice-cold ethanol series. DNA-BAC probes were denatured at 80°C, pre-hybridized at 37°C, and incubated with the sample overnight at 37°C. After stringency washes, digoxigenin was detected with anti-digoxigenin-FITC (1:10, Millipore). RNA FISH slides were then stained for immunofluorescence with anti-TOPBP1 (1:50, Abcam) and anti-γH2AX (1:100, Millipore). Samples were analyzed on an Olympus IX70 inverted microscope and computer-assisted (DeltaVision) CCD camera (Photometrics) was used to capture images (processed with ImageJ and Photoshop software).