Lsm 780 inverse laser scanning microscope

Frozen OCT sections from the postprogression biopsy (Core 2 OCT block for serial sectioning) and the PDX sample treated with 450 mg/kg rucaparib for 2 weeks, both from patient 5 with the germline RAD51C mutation, as well as an unrelated control PDX were thawed, fixed in 4% paraformaldehyde, and pretreated with a SPOT-Light Tissue Pretreatment kit (Invitrogen). Briefly, sections were incubated in pretreatment solution for 15 minutes at 95°C, washed in PBS, and incubated with enzyme for 10 minutes at room temperature. Sections were dehydrated, incubated with denaturation buffer (70% formamide, 2× SSC, pH 7.0–8.0) for 5 minutes at 73°C, dehydrated, and incubated with prepared RAD51C probe (as per the manufacturer's instructions; Empire Genomics) for 24 hours at 37°C. Sections were then washed with WS1 (0.4× SSC/0.3% NP-40) for 2 minutes at 73°C, followed by a wash with WS2 (2× SSC/0.1% NP-40) for 1 minute at room temperature. Nuclei were counterstained with Hoechst 33342 (NucBlue Live ReadyProbes Reagent; Thermofisher Scientific) and coverslipped with fluoromount G (SouthernBiotech). Sections were imaged using an LSM 780 inverse laser scanning microscope (Zeiss) and captured with an LSM T-PMT detector (Zeiss).

Cells were treated with DMSO or 10 μmol/L rucaparib for 24, 48, or 72 hours. Cells were fixed with either 4% paraformaldehyde or methanol, permeabilized with 0.2% TritonX-100, blocked with blocking buffer (1% BSA, 2% FCS or 1% BSA, 3% milk, 2% goat serum in PBS) and incubated with rabbit anti-RAD51 (ab133534 1:250; ab11055 1:400; Abcam) and either mouse anti-Geminin (ab104306 1:100; Abcam) or mouse anti-γH2AX (ab26350 1:400; Abcam) antibodies. For RAD51 foci formation with geminin staining, anti-rabbit 647, and anti-mouse 546 Alexa Fluor secondary antibodies (1:800; Invitrogen Molecular Probes) were used. Nuclei were counterstained with Hoechst 33342 (NucBlue Live ReadyProbes Reagent; Ther-mofisher Scientific). Cells were imaged using an LSM 780 inverse laser scanning microscope (Zeiss) and captured with a LSM T-PMT detector (Zeiss). At least 194 cells from four fields of view and two independent experiments were counted. Cells with ≥5 RAD51 foci/nucleus were scored using CellProfiler (version 2.2.0, Broad Institute). For RAD51 and γH2AX foci formation assay, anti-rabbit 488 and anti-mouse 594 Alexa Fluor secondary antibodies (A32731 1:500; A-11032 1:500; Invitrogen Molecular Probes) were used. Nuclei were counterstained with DAPI (Sigma). Cells were imaged using Leica DM 1000 LED at 40×.