Alexa fluor 594 goat anti mouse igg1

Cells of each group were seeded at a density of 1.2 × 10⁴ cells in expansion medium. After 24 h, the medium was replaced by differentiation medium. After fixation with ice-cold methanol, slides were washed and incubated in blocking buffer consisting of PBS with 1.5% FCS and 0.25% TritonX (Carl Roth GmbH, Karlsruhe, Germany) for 1 h at room temperature. After washing with TBS-T buffer (100 mM Tris and 60 mM NaCl in distilled water, 1 ml Tween20 per 1 L; pH 7.6), slides were covered with primary antibodies (anti-desmin (AB-1 (D33), Thermo Fisher Scientific, Runcorn, Cheshire, UK), anti-alpha-sarcomeric actinin (EA-53, Abcam), anti-MEF2 (MEF2A, B-4, Santa Cruz Biotechnology), anti-myosin heavy chain 2 (MYSN02, MyHC2, Thermo Fisher Scientific)) and diluted 1:50 in blocking buffer for 1 h at room temperature. As secondary antibody, Alexa Fluor 594 goat-anti-mouse IgG1 (Invitrogen, Karlsruhe, Germany) was used at 1:200 for 30 min at room temperature. Probes were counterstained with DAPI 1:1000 (Diamidine-phenylindole-dihydrochloride, Applied Science/Roche, Indianapolis; Indiana, USA) for 5 min. Slides were subsequently analysed and digitally photographed with a fluorescence microscope (IX83, cellSens software, Olympus, Hamburg, Germany). L6-Mb served as the positive control. An isotype control was performed using mouse IgG1 (BD Biosciences).

Hybridization chain reaction (HCR) and Whole Mount Immunohistochemistry were done in accordance with previous described methods [42 (link), 61 (link)]. The HCR probes transcripts were synthesized by Molecular Instruments for ret, NM_181662.2; gfra1a, NM_131730.1; oprl1, NM_205589.2; oprd1b, NM_131258.4; etv1, XM_005157634.4; elavl3, NM_131449. The following primary antibodies were used: goat polyclonal IgG anti-Choline Acetyltransferase (ChaT, Millipore Sigma, AB144P, 1:500), rabbit polyclonal IgG anti-5-HT (serotonin, Immunostar, 20080, 1:250), mouse monoclonal IgG2b anti-HuC/D (Invitrogen Thermo Fisher, A-21271, 1:250), Mouse monoclonal IgG1 anti-Phox2b (B-11, Santa Cruz Biotechnology, SC-376997, 1:250). The following secondary antibodies were used from Invitrogen: Alexa Fluor 488 donkey anti-goat IgG (A-11055, 1:600), Alexa Fluor 405 goat anti-rabbit IgG (A-48254, 1:600), Alexa Fluor 647 goat anti-mouse IgG2b (A-21242, 1:600), Alexa Fluor 594 goat anti-mouse IgG1 (A-21125, 1:600). High content semi-automated confocal imaging and processing was done as described above.

The 2C11 affinity purified anti-PI(4,5)P2 antibody was from Echelon (Salt Lake City, UT, USA) product number Z-P045, lot number XCM042523-23, and used at 1:200 dilution in blocking buffer (see IF methods for blocking buffer). The affinity-purified anti-PI(3,4,5)P3 antibody was also from Echelon product number Z-G345, lot number ML120516-23, and used at 1:200 dilution in blocking buffer. The anti-3X FLAG M2 monoclonal antibody was from Sigma (St. Louis) and used at 1:1000 dilution in blocking buffer. For colocalization of FLAG and PIP2 in the same chamber, mouse FLAG-primary M2 antibodies were detected with 1:1000 PBS-diluted Invitrogen Alexa-Fluor 594 goat anti-mouse-IgG1, lot number 2566384, while 2C11 anti-PIP2 primary antibodies were detected with 1:2000 PBS-diluted Invitrogen Alexa-Fluor 488 goat anti-mouse-IgM, lot number 1896382. The anti-actin antibody was from Cell-Signaling (product number 8H10D10) and was used at 1:5000 dilution in 1X TBST. The secondary antibody in the Westerns was Promega anti-mouse HRP conjugate (product number W402B) and was used at 1:10,000 dilution in 1X TBST.

Two days before IR treatment, 5 × 10⁴ (MCF-7) or 2.5 × 10⁴ (MCF10A) cells per well (1.8 cm²) were seeded as duplicates in chamber slides (LabTek®, Nunc, Roskilde, Denmark). The addition of SVA was performed 24 h later. After fixation with 2% formaldehyde and permeabilization with 0.25% triton-X 100 (both Sigma Aldrich Chemie GmbH, Munich, Germany), the cells were consecutively incubated for 60 min with the anti-γH2AX antibody (1:500, clone JBW301, Merck Millipore) and Alexa Fluor 594 goat anti-mouse IgG1 (1:400, Molecular Probes®/Life Technologies, Darmstadt, Germany) for 30 min. The slides were mounted with Vectashield® containing anti-4′,6-diamidino-2-phenylindole (DAPI; Vector Laboratories, Inc., Burlingame, CA). The foci were visualized with an Eclipse TE300 inverted microscope (Nikon, Tokyo, Japan). At the magnification of 1000x, the foci of 50 cells per chamber were counted; two chambers per SVA concentration and irradiation dose were analysed.

Twenty-four hours before IR, 35,000 pADSCs were seeded in duplicate in chamber slides (LabTek^®, Nunc, Roskilde, Denmark). After fixation with 2% formaldehyde and permeabilisation with 0.25% Triton X-100 (both Sigma Aldrich Chemie GmbH), the cells were consecutively incubated 60 min with anti-γH2AX antibody (1:500, clone JBW301, Merck Millipore) and Alexa Fluor 594 goat anti-mouse IgG1 (1:400, Molecular Probes^®/Life Technologies, Darmstadt, Germany) for 30 min. The slides were mounted with Vectashield^® containing anti-4′,6-diamidino-2-phenylindole (DAPI; Vector Laboratories, Inc., Burlingame, CA, USA). The foci were visualised with an Eclipse TE300 inverted microscope (Nikon, Tokyo, Japan). At a magnification of 1000×, the foci of 50 cells per chamber were counted; two chambers per IR dose.

Directly after irradiation, 1 × 10⁴ cells per well (1.8 cm²) were seeded in duplicate in chamber slides (LabTek®, Nunc, Roskilde, Denmark) and incubated for 24 h. After fixation with 2% formaldehyde and permeabilisation with 0.25% triton-X 100 (both Sigma Aldrich Chemie GmbH, Munich, Germany) the cells were consecutively incubated 60 min with anti-γH2AX antibody (1:500, clone JBW301, Merck Millipore) and Alexa Fluor 594 goat anti-mouse IgG1 (1:400, Molecular Probes®/Life Technologies, Darmstadt, Germany) for 30 min. The slides were mounted with Vectashield® containing anti-4′,6-diamidino-2-phenylindole (DAPI; Vector Laboratories, Inc., Burlingame, CA). The foci were visualised with an Eclipse TE300 inverted microscope (Nikon, Tokyo, Japan). At a magnification of 1000×, the foci of 50 cells per chamber were counted; two chambers per irradiation. The extra yield (∆Y) was calculated as the difference between irradiated samples and the individual 0 Gy control value of residual foci as a function of dose and plotted in a graph. Linearisation was performed as described by Barendsen [30 (link)]. RBE values, referred to as RBE_{(foci 24 h)} in the text, were calculated with respect to LDR X-rays via the α value with reference to Franken et al. [31 (link), 32 (link)]. Fits to the data points using the eq. F(D) = αD + βD² yielded β values of zero.