Alexa secondary antibodies

NTN1 recognizing antibody (CH23002) was obtained from Neuromics. Monoclonal antibody against CD133 (W6B3C1, MACS Miltenyi), polyclonal antibody against nestin (3579, Millipore) and polyclonal antibody against Sox2 (Cell Signaling) were used to recognize the stemness markers of GBM cells. Anti hemaglutinin antibodies from Covance (HA-11) and from Sigma-Aldrich (clone 3 F10) were used to recognize tagged NTN1. The tumor vasculature was stained with mouse CD31 recognizing antibody from R&D Systems. Notch2 extracellular domain (25–255) and intracellular domain (D67C8) recognizing antibodies were obtained from Santa Cruz Biotechnology and Millipore respectively. Antibody recognizing cleaved Notch2 (D1733) was obtained from Immunoway. Intracellular domain of Jagged1 (C-20) recognizing polyclonal antibody was obtained from Santa Cruz Biotechnology. Human specific lamin A/C antibody (Novus Bio) was used to distinguish human tumor cell in mice xenografts. The fluorochrome conjugated Alexa secondary antibodies used in immunofluorescence microscopy were purchased from Invitrogen. The nuclei were visualized with Hoechst 44432 (Invitrogen). Recombinant human EGF, basic FGF and NTN1 proteins were obtained from R&D Systems. Growth factor reduced Matrigel was obtained from Corning.

Muscles were cut into 10 μm muscle cross-sections and mounted on glass slides to be stained, as described below. Trichrome staining was conducted by the Histology Core Facility at the University of Ottawa following Masson’s Trichrome protocol⁴⁸ . For immunofluorescence analyses, muscle cross-sections were fixed with 2% paraformaldehyde (PFA) for 7 minutes, permeabilized using 0.1% Triton X-100 (Thermo Fisher Scientific, Waltham, MA) for 10 minutes at room temperature, blocked using either the M.O.M Blocking kit (Pax7; Vector Labs, Burlingame, CA), DAKO Serum-Free Protein Block (PDGFRα; Agilent, Santa Clara, CA), or standard serum block (Laminin, Perilipin, and p-NFkB; PBS with 5% NGS, 1% BSA, and 0.1% Triton X-100). Sections were incubated with primary antibody overnight at 4 °C (mouse anti-Pax7, DSHB, neat; rabbit anti-MyoD, Abcam, 1:200; rat anti-Laminin, ThermoFisher Scientific, 1:200; rabbit anti-PDGFRα, R&D Systems; 1:250; rabbit anti-phospho-NFĸB, Cell Signaling, 1:200; Perilipin, Cell Signaling, 1:100). Appropriate Alexa secondary antibodies (Invitrogen, Carlsbad, CA) were used for detection of each primary antibody (1:300), while nuclei were counterstained using 4,6-diamidino-2-phenylindole (DAPI, 1:10000).

For in vivo studies, fixed frozen brains were sliced at 40 μm thickness. The slices were immunologically stained using the free-floating method. The primary antibodies used are listed in Supplementary Table 2. For staining with CD3 and CD4 antibodies, we took an additional step for antigen retrieval using 10 mM citrate buffer at 100 °C for 40 min. Fluorescent staining with Alexa secondary antibodies (Invitrogen) were used. The cells were visualized using a confocal laser microscope (LSM700, Zeiss). For 3, 3′-Diaminobenzidine-nickel (DAB-Ni) staining, biotinylated secondary antibody and ABC Elite kit (Vector Labs) was performed followed by 0.02% DAB, 0.6% nickel ammonium sulfate, and 0.0045% H₂O₂ incubation. For DAB-Ni and HE staining, we observed the samples and took pictures with BZ-X700 (Keyence, Osaka, Japan). For DAB-Ni staining, dead cells and debris are shown in brown, and positive signals are stained in dark blue and black. The number of immunoreactive cells was quantified in every 18th section throughout the grafts and surrounding tissue and corrected by using the Abercrombie method.

Adult testes were collected and fixed in 4% paraformaldehyde overnight and embedded in paraffin. 6 µm sections were cut for HA and γH2AX immunostaining. Sections were subjected to antigen retrieval using steam vapor for 30 minutes in antigen unmasking solution (Vector Lab) and then permeabilized for 10 minutes at room temperature in 0.1% triton-X. Sections were blocked 30 minutes at room temperature in 10% normal donkey serum, 2% BSA and 0.1M glycine (Sigma). Primary antibody incubation was done overnight at 4°C in the blocking buffer. Appropriate Alexa secondary antibodies (Invitrogen) (1∶1000) were used. Hoechst 33342 (5 µg/ml) (Sigma) was used to stain DNA. Leica TCS SP5 confocal microscope was used to acquire all images. Photoshop was used for cropping and other modifications that were equally performed on control or experimental samples.

Coverslip covered with cells seeded in six-well plates, TNF-α treated 36 hours, when the fused cells grew to 95% -100%, was removed from the incubator, the cell culture medium was discarded. Cells were fixed with 4% paraformaldehyde for 20 min on the ice, 0. 1% of Triton X - 100 at room temperature for 5 min, and blocked with 1% BSA for 30min. Then samples were incubated with primary antibodies E-cadherin (1:200), N-cadherin (1:200), vimentin (1:50) which were used as described in western blot at 4℃ over night, and were incubated avoid light with Alexa secondary antibodies (Invitrogen, 1:400) at room temperature for 1 h. After washing, wells were covered with DAPI (Invitrogen). Preserved void light at 4℃ for microscopic examination.

Cells were fixed in 4% paraformaldehyde/PBS, permeabilized in 0.1% Triton X-100/PBS, washed in 1% BSA/PBS, blocked with 4% BSA/PBS, and stained with rabbit polyclonal anti-p65 (1:200, sc-8008, Santa Cruz Biotechnology). Cells were incubated with Alexa secondary antibodies (Invitrogen) and nuclei counterstained with propidium iodide or 4′,6-diamidino-2-phenylindole (DAPI). Cells were analyzed using a Confocal System TCS SP5 (Leica).
Cell uptake of St-PGA-CL-BDMC was assessed by live fluorescence imaging in microscopy chambers (1 µ-slides) of cultured cells treated with 10 µM St-PGA-CL-BDMC for 6 h. After stimulation, cells were washed with PBS-BSA 0.1% and then incubated for 20 min with the lysosomal marker LysoTrackerTMGreen DND-26 (Invitrogen). After washing with PBS-BSA 0.1%, cells were left in culture medium and placed in the microscope culture chamber (37 °C, 5% CO₂) and analyzed using the same confocal system.