Anti mouse igg alexafluor 488

The following primary antibodies were used in this study: anti-CEP128 [Abcam, ab118797; immunofluorescence (IF) 1:500, western blotting (WB) 1:1000], anti-γ-tubulin (Merck, GTU88; IF 1:1000), anti-p15 (Santa Cruz Biotechnology, sc-271791; WB 1:1000), anti-centrin (Millipore, A302-479A; IF 1:1000), anti-KLC1 (Abcam, ab187179; IF 1:500), anti-HSP90 (BD Biosciences, 610419; WB 1:1000) and anti-β-actin (Santa Cruz Biotechnology, sc-47778; WB 1:1000). The following secondary antibodies were used: anti-mouse IgG Alexa Fluor 488 (Molecular Probes, 1:1000), anti-rabbit IgG Alexa Fluor 555 (Molecular Probes, 1:1000), anti-mouse IgG HRP (Promega, WB 1:10,000) and anti-rabbit IgG HRP (Promega, WB 1:10,000).

For visualisation of ovalbumin-Alexa Fluor 647 sections were imaged on a confocal laser scanning microscope (LSM880, Zeiss). To determine the location of tracer with regards to the walls of blood vessels, sections were incubated with Rat Endothelial Cell Antibody (1:100; RECA-1; AB9774 Abcam, Cambridge, United Kingdom) followed by a secondary antibody (1:400; anti-mouse IgG Alexa Fluor 488; Molecular Probes, Life Technologies, New York, USA) and with actin α-smooth muscle antibody (1:400; SMA-Cy3; Sigma-Aldrich, St. Louis, Montana). Images were acquired in a z-stack scan mode, with interval of 0.5 μm, as a 4 line average, pixel dwell 0.76 μs, using a plan-apochromat 40x/1.3 oil DIC UV-IR M27 objective. Image dimensions were x: 1024, y: 1024, z: 36 μm; 8-bit.

The glass slides were washed twice for 10 min in tris–phosphate buffered saline, and then in 50% ethanol for cellular permeabilization. After application of 15% normal donkey serum (NDS) blocking solution, the slides were incubated overnight with 1:100 Rat Endothelial Cell Antibody (RECA-1, Abcam, Cambridge, United Kingdom) in 4% NDS. The secondary antibody, 1:400 anti-mouse IgG Alexa-Fluor^®-488 (Molecular Probes, Life Technologies, New York, USA) was then applied. This was followed by anti-actin α-smooth muscle antibody at 1:400 dilution (SMA-Cy3, Sigma-Aldrich, St. Louis, Montana). Primary and secondary controls were established to exclude autofluorescence. The slides were cover-slipped with fluorescent mounting medium (DAKO, NSW, Australia).

The cells were washed and fixed as follows. For 53BP1 staining, the cells were treated with CSK buffer (10 mM PIPES, 300 mM sucrose, 100 mM NaCl and 3 mM MgCl₂) for 1 min, then with 0.5% Triton X-100 in CSK buffer for 1 min, and finally with 4% PFA in PBS for 10 min at room temperature; for Cyclin A, the γH2AX and CldU staining cells were treated with 4% PFA for 10 min and 0.3% Triton Χ-100 in PBS for 5 min at room temperature; the S9.6 staining cells were treated with methanol for 10 min on ice and 0.5% Triton X-100 in PBS for 5 min at room temperature. The samples were blocked with 3% BSA/10% foetal bovine serum in PBS. Primary antibodies were mouse anti-phospho-Histone H2AX (Ser139) (JBW301, Millipore 05-636, 1:1000), rabbit anti-53BP1 (Bethyl A300-272A, 1:30,000), mouse anti-Cyclin A (6E6, Thermo Scientific MS1061, 1:50), rat anti-CldU (BU1/75, AbD Serotec OBT0030G, 1:250) and mouse anti-RNA/DNA hybrid (S9.6, gift from Professor Richard Gibbons, hybridoma supernatant 1:100). The secondary antibodies were anti-mouse IgG AlexaFluor 488 and anti-rabbit IgG AlexaFluor 555 MolecularProbes). DNA was counterstained with 4,6-diamidino-2-phenylindole (DAPI) and images acquired as above. For the quantification of nuclear S9.6 intensity, ImageJ was used to generate nuclear masks based on DAPI staining and mean S9.6 fluorescence intensities per pixel were quantified per nucleus.