Goat anti mouse alexa fluor 546

Dissected Drosophila tissues or C2C12 cells grown on coverslips were rinsed in PBS (Lonza 17-512F) and fixed in 4% paraformaldehyde (Sigma P6148) for 20 min at room temperature. Following fixation, the samples were washed four times (×10 min) in PBS and blocked with blocking buffer: 5% normal goat serum (NGS; Abcam AB7681) in PBS with 0.1% TritonX-100 (PBST). The samples were incubated with primary antibody overnight at 4 °C, washed four times (x10 min) with 0.1% PBST, incubated with secondary antibody for 2 h at room temperature followed by 0.1% PBST washes. Samples were mounted onto slides using either ProLong^® Gold Antifade mounting reagent (Invitrogen P36930) or Fluoroshield (Sigma F6057).
Primary and secondary antibodies were prepared in blocking buffer.
Primary antibodies: Cy3-conjugated anti-HRP (1:100, Jackson ImmunoResearch 123-165-021); Rabbit anti-FLAG (1:500, Sigma F7425); Mouse anti-hnRNPM 2A6 (1:100; Santa Cruz sc-20001); Rabbit anti-MATR3 (1:500; Abcam ab151714)
Secondary antibodies: Goat anti-rabbit Alexa Fluor 488 (1:1000; Invitrogen A11008); Goat anti-mouse Alexa Fluor 546 (1:1000; Invitrogen A11030)

For doing western blots in Drosophila, the following primary antibodies were used: anti-FUS (Bethyl Laboratories; A300-302A, 1:2000), anti-MBNL1 (Millipore; MabE70, 1:2000), and anti-mbnl1 (Invitrogen; MA1-16967, 1:1000). DyLight fluorescent secondary antibodies (Thermo Fisher Scientific) were used at a concentration of 1:10,000
For the Drosophila NMJ analysis, the following primary antibodies were used: Alexa Fluor 488-conjugated goat anti-HRP (Jackson Immuno Research; 123-545-021, 1:200) and mouse anti-DLG 4F3 (DSHB, 1:100). The following secondary antibodies were used: Alexa Fluor 647-conjugated anti-Phalloidin (Invitrogen; A22287, 1:250) and goat antimouse Alexa Fluor 546 (Invitrogen, A11030, 1:500).
The following primary antibodies were used: anti-G3BP1 (Protein Tech; 13057-2-AP, 1:2000) and anti-HA7 (Sigma, H3663, 1:500). Alexa-Fluor fluorescent secondary antibodies (Invitrogen) were used at a concentration of 1:500 for doing Western blots in HEK293 and N2a cell lines.
The following primary antibodies were used for doing immunoblotting in primary neuronal cells: anti-MAP2 (EMD Millipore; AB5622, 1:500), anti-FUS (Proteintech; 11570-1-AP, 1:200), anti-TIA1 (Santa Cruz; SC-1751, 1:250), anti-G3BP1 (Proteintech; D5444, 1:100), anti-MBNL1 (Millipore; MABE70, 1:100), and anti-NEFL (Novus Biologicals; NB300-222, 1:300).

Cells were fixed with 4% paraformaldehyde in PBS (pH 7.4) for 30 min, permeabilized and blocked with 0.1% Triton X-100 (Sigma-Aldrich, St. Louis, MO) and 10% normal goat serum in PBS for 1 h, washed with PBS, and incubated with primary antibodies (polyclonal Ack1 1:100; monoclonal CAMKII-α 1:100) for 2 h. After being washed with PBS, cells were incubated with both secondary antibodies, namely goat anti-mouse-Alexa Fluor-546 or goat anti-mouse-Alexa Fluor-488 (Invitrogen, Carlsbad, CA), for 1 h. Coverslips were washed with PBS and mounted on glass slides. All procedures were carried out at room temperature [29 (link)].

ASF-2 cells were grown in DMEM (Lonza) with 10% fetal bovine serum (Thermo Scientific), 100 U/mL penicillin and streptomycin (Lonza) at 37°C, 5% CO₂ and 95% humidity. ASF-2 cells of passage 10 were detached from a tissue culture flask by trypsination with Trypsin EDTA (Lonza) and spun down at 600 g for 6 minutes. Cells were then resuspended in growth medium and 15.000 cells/well were seeded out in an ibiTreat μ-Slide VI 0.4 (Ibidi) and grown overnight. The next day the cells were rinsed with PBS, fixed in 4% PFA for 15 min., permeabilised with 0.025% Triton X100 (Sigma-Aldrich) for 10 min. and blocked with 2% BSA in PBS (BSA-PBS) for 1 hour at room temperature. The cells were incubated with 5 μg of purified LOB7 in 2% BSA-PBS or 100 μl of a 1:100 dilution of V9 antibody in 2% BSA-PBS (Sigma Aldrich) pr. well for 1 hour at room temperature. Visualisation of LOB7 was accomplished by incubation with a 1:100 dilution of Goat-anti-Rabbit Alexa Fluor 488 (Invitrogen, USA). V9 was visualised by a 1:100 dilution of Goat-anti-Mouse Alexa Fluor 546 (Invitrogen). Cell nuclei were stained with Vectashield Mounting Medium with DAPI (Vector Labs). Fluorescent images were obtained with a Leica DMI3000 B inverted microscope (Leica Microsystems).

Resting B cells and CH12 were fixed with 4% paraformaldehyde (Sigma-Aldrich) and permeabilized with PBST (0.1% Tween 20 in PBS 1×) containing 0.2% IGEPAL CA-630 (Sigma-Aldrich). Cells were stained with antibodies against Pdap1 (Sigma-Aldrich), rat anti-mouse IgM-PE (BD Biosciences), and goat anti-rabbit Alexa Fluor 488 (Abcam). Cells were incubated with permeable nuclear dye (Hoechst dye 33258, Thermo Fisher Scientific) and transferred onto slides for imaging.
iMEFs infected with either empty pMX vector (EV) or pMX-Pdap1-3xFlag construct were grown on coverslips overnight. Upon fixation with 4% paraformaldehyde and permeabilization with 0.5% Triton X-100, cells were stained with mouse anti-Flag M2 (Sigma-Aldrich), goat anti-mouse Alexa Fluor 546 (Invitrogen), and Hoechst dye 33258. Images were acquired using inverted LSM700 AxioObserver laser scanning confocal microscope (Zeiss), with Plan-Apochromat 63×/1.40 Oil Ph3 objective for primary B cells and CH12, and EC Plan-Neofluar 40×/1.30 Oil Ph3 objective for iMEFs.

Cells were plated in 35-mm glass-bottomed dishes from Mattek (MA, USA) and allowed to attach overnight. AZD6738 and KU-0060648 were added 1 h before irradiation. Samples were fixed in 10% neutral buffered formalin, permeabilized in 0.2% Triton X-100, and treated with DNaseI from Roche (West Sussex, UK). Cells were blocked in 1% BSA, 2% FCS in PBS before staining using antibodies for γH2Ax (S139) clone 20E3 from Cell Signaling (MA, USA), or RAD51 clone 14B4 from Genetex (CA, USA) with goat anti-rabbit AlexaFluor 488 or goat anti-mouse AlexaFluor 546 as secondary antibodies from Invitrogen (Paisley, UK). Nuclei were counterstained with DAPI. Samples were imaged using a Zeiss LSM710 inverted confocal microscope (Jena, Germany). Individual nuclei were scored manually for normal morphology, nuclear fragmentation such as micronuclei, or gross lobular irregularities. Nuclei were also scored for γH2Ax foci. >5 γH2Ax were scored as positive. Pan-nuclear γH2Ax staining was also scored if γH2Ax foci numbers were unquantifiable. RAD51 and γH2Ax foci were also quantified by automated image quantification using Cell Profiler v2.2 (Broad Institute, CA, USA). Nuclei were segmented and counted based on DAPI staining, RAD51 and γH2Ax were counted and expressed as average foci per nucleus. Quantification of each independent experiment included approximately 100–300 nuclei.