A21422

Human neutrophils were isolated, seeded (200,000/well) in 24-well plates on glass coverslips and activated to undergo NET formation as described above. After fixation with 2 % PFA (Roth) over night, cells were permeabilized 0.1 % TritonX (Merck) and incubated with 5 % FCS (Biochrom) to block unspecific antibody binding. Subsequently, cells were stained using monoclonal anti-human MPO (IgG, mouse) as primary antibody (Abcam, ab25989, 1:500) and polyclonal anti-mouse Alexa 555 (IgG, goat) as secondary antibody (Life technologies, A21422, 1:2000). Neutrophil DNA was stained with 1.62 μM Hoechst (Sigma-Aldrich) as described above. After the staining procedure, cells were stored protected from light at 4°C. Representative confocal fluorescence images were obtained with the olympus IX83 inverted microscope (software: Olympus Fluoview Ver.4.2, Olympus) and recorded 60x magnified (UPlanSApo 1.35 oil, Olympus). All pictures were recorded at equal exposure times for MPO to ensure comparability.

Oocytes were fixed for 30–60 min at 37 °C in 100 mM HEPES (pH7; titrated with KOH), 50 mM EGTA (pH7; titrated with KOH), 10 mM MgSO₄, 2% formaldehyde (methanol free) and 0.2% Triton X-100. Oocytes were left in PBS (phosphate-buffered saline) with 0.1% Triton X-100 overnight at 4 °C. F-actin staining was carried out in PBS with 0.1% Triton X-100 and 3% bovine serum albumin with Alexa-Fluor-488–Phalloidin (Molecular Probes, A12379; 1:100). DNA was stained with 5 mg ml⁻¹ Hoechst 33342 or DAPI (Molecular Probes). As primary antibodies, we used mouse Cyclin B (Abcam, ab2949; 1:100). As secondary antibodies, we used Alexa-555-labelled goat anti-mouse (Life technologies, A21422; 1:500).

Whole mount immunofluorescence was performed as described in Ref.^{69 (link)}. Following FISH, a rabbit polyclonal anti-TH primary antibody^{22 (link)} was used at 1:500 dilution and detected with an anti-rabbit Alexa555-conjugated secondary antibody (2 µg/ml, Life Technologies A-21428). For double FIHC (Tg(top:dGFP)^w25 and Tg(7xtcf-Xla.siam:gfp)^ia4 embryos), a chicken anti-GFP antibody (5 µg/ml; Invitrogen) was combined with a polyclonal rabbit anti-TH antibody^{22 (link)}. Primary antibodies were detected with an anti-chicken Alexa488 antibody (2 µg/ml; Life Technologies A11039) and an anti-rabbit Alexa555 antibody (2 µg/ml, Life Technologies A21430). Following FISH for sox2 and sox3, primary mouse-anti-Sox2 antibody (2.5 µg/ml, Abcam, ab171380) was used and subsequently detected using an anti-mouse-Alexa633 (2 µg/ml, Life Technologies A21050) antibody. Following EdU detection, the Sox2 antibody was detected using an anti-mouse-Alexa555 secondary antibody (2 µg/ml, Life Technologies A21422).

Cells were cultured on gelatin (0.1%) coated coverslips in 12-well plates, treated with CPT (1 μM, 1 hr) and then recovered for 2 hr in medium without CPT. Cells were collected at indicated time points and fixed in 4% PFA for 20 min, permeabilized in PBS with 0.5% Triton X 100 (EMD, TX1568-1) and blocked in 1% BSA in 1× PBS (P0195, Teknova, Hollister, CA, United States) for 30 min. Human prostate cells were then incubated in anti-Phospho (Ser139) H2AX (NBP1-64745, 1:500, Novus Biologicals, Littleton, CO, United States), anti-MYC-tag (clone 4A6, Millipore, 1:300), anti-RAD51 (PC130, 1:500, Calbiochem, United Kingdom), or anti-53BP1 (NB 100-904, Novus Biologicals, 1:500) primary antibodies followed by Alexa Fluor 555 or Alexa Fluor 488 labeled anti-mouse or anti-rabbit secondary antibodies (1:500) (A-21422, A-1101, Life Technologies). Mouse prostate epithelial cells were incubated in anti-phospho ATM (ab36810, 1:500, Abcam, United Kingdom), anti-Rad51 (Abcam, ab88572, 1:100), anti-γH2AX (Abcam, ab26350, 1:1000), and anti-SPOP (developed in the Rubin lab, 1:300). Pictures were taken on a Zeiss LSM 510 Laser Scanning Confocal Microscope.

Cells were fixed with 4% paraformaldehyde for 15 min at RT followed by three washes with PBS. Cells were permeabilized with 0.25% Triton X-100 in PBS, and blocked for 1 hr in blocking buffer at RT (1% BSA, 300 mM Glycine, 0.1% Gelatin, 4% Donkey Serum in TBST). After blocking, cells were incubated with primary antibodies diluted in blocking buffer overnight at 4 °C. The day after cells were washed three times (5 min each) with 0.05% Tween-20 in PBS. The following primary antibodies were used: Tau-46 (Invitrogen, 36400, 1:1000), MAP2 (Millipore, AB5622, 1:1000), and anti-his (Thermo Scientific, MA1-21315, 1:1000). Secondary antibodies (Life Technologies, A21422, A21206, 1:1000) were incubated for 1 hr at RT, washed three times with 0.05% Tween-20 in PBS and imaged with an Olympus IX71 Microscope or an Olympus Fluoview 1000 Spectral Confocal.

IF staining was performed according to standard protocols [31 (link), 32 (link)]. The primary antibodies used were Ki-67 (1:200, #ab16667; Abcam) and anti-α-SMA (1:200, #ab240654; Abcam). The secondary antibodies used were donkey anti-rabbit Alexa Fluor 488 (1:200, #A21206; Thermo Fisher) and goat anti-mouse Alexa Fluor 555 (1:200, #A21422; Thermo Fisher). Nuclei were stained with 4′,6‐diamidino‐2‐phenylindole (DAPI, Life Technologies) for 5 min. Images were obtained using an inverted fluorescence microscope (Leica). Ki-67 positive cells and Ki-67/α-SMA double-positive cells were counted and averaged for quantitative analysis.