Anti mouse alexa fluor 555 conjugated secondary antibody

Cells plated on dishes containing glass coverslips coated with fibronectin were washed in 3 × 2 mL PBS and fixed in 2% (w/v) paraformaldehyde in PBS pH 7.2 for 10 min. Cells were washed for 3 × 5 min in PBS followed by permeabilization in PBS, 0.15% (v/v) Triton for 10 min. Cells were blocked in FX Image-iT^™ Signal Enhancer (Thermo Fisher) for 30 min and then stained with the anti-γ-H2AX antibody at a 1:100 dilution in PBS, 2% (w/v) BSA for 1 h. After washing in PBS, 2% (w/v) BSA, the cells were incubated with anti-mouse Alexa Fluor 555-conjugated secondary antibody (Thermo Fisher) at a 1:500 dilution in PBS, 2% (w/v) BSA for 45 min. Cells were washed in PBS, stained with SYTO^™ 13 green fluorescent nucleic acid stain (400 nM, Thermo Fisher) in PBS and incubated at room temperature in the dark for 1 h prior to imaging.

Immunofluorescence labeling for Delta D was conducted on regenerating neuromasts of 4 dpf larvae. Larvae were fixed overnight at 4 °C in 4 % formaldehyde in phosphate-buffered saline solution (PBS). On the following day, the larvae were washed for 5 min in PBS with 1 % Tween-20 (PBST) and placed for 2 hr in blocking solution containing PBS, 10 % NDS, 0.5 % Triton X-100, 2 % bovine serum albumin, and 1 % dimethylsulfoxide. The primary antibody, murine anti-Delta D (1:100; zdd2, Abcam) [47 (link)], was diluted in fresh blocking solution and incubated with the larvae overnight at 4 °C. Larvae were washed for 2 hr with PBS containing 0.1 % Triton X-100. Pre-adsorbed AlexaFluor 555-conjugated anti-mouse secondary antibody (Invitrogen, Molecular Probes) was applied overnight at 4 °C at a dilution of 1:200 in blocking solution. The pre-adsorption step was introduced to increase the specificity of the antibody in order to minimize background signal. Larvae were then washed for 2 hr in PBS containing 0.1 % Triton X-100 and stored at 4 °C in VectaShield.

Wild-type (WT) and Polη mutants were overexpressed by integration of the GAL promoter upstream of respective ORF, except the Polη-S14A mutant, for which the constitutive strong GPD or ADH promoter was utilized. To compare nuclear accumulation with and without break induction, isogenic strains with or without the P_GAL-HO allele were prepared. Cells were fixed with formaldehyde (final 3.7%) and spheroplasts were prepared through zymolyase treatment. The spheroplasts were subsequently applied on poly-L-lysine precoated slides, and fixed with ice-cold methanol followed by acetone, before blocking (3% BSA in 1× PBS). Cells were then stained with either anti-FLAG or anti-Myc primary antibody at 4° overnight. After 10 washes with blocking solution, the slides were incubated in the dark for 1 hr with Alexa Fluor 555-conjugated anti-mouse secondary antibody (Invitrogen, Carlsbad, CA). Finally, the slides were washed 10 times with blocking solution, once with 1× PBS, and then mounted with ProLong Gold or ProLong Diamond including DAPI. An untagged strain and secondary antibody alone were included as controls. Image overlays were generated with the Openlab software (Improvision) or with ImageJ. The experiment was repeated at least twice and at least 200 cells of each sample from individual experiments were assessed.