Cy3 conjugated anti mouse igg

Control and treated ECFCs were grown on coverslips in EGM-2, fixed and permeabilized according to routine immunocytochemistry methods [18 (link)]. The anti-human primary antibodies used were: anti-uPAR R3 (1:40, rabbit polyclonal, Santa Cruz, CA, USA), anti-caveolin-1 (1:400; Sigma-Aldrich), anti-GM3 (1:100, mouse monoclonal Ab, Cosmo Bio, DBA Italia, Milano, Italy) Cholera toxin-beta subunit (CTB; 10 μg/ml; Sigma-Aldrich) was used to study GM1 distribution. The secondary antibodies used for single and double immunostainings were: CY3-conjugated antimouse IgG (1:800; C2181; Sigma-Aldrich) and FITC-conjugated anti-rabbit IgG (1:800; F-4151; Sigma-Aldrich). For nuclear staining, samples were incubated with DAPI (2 μg/ml), for 15 min. The mounting procedure and the confocal analysis were run as previously described [8 (link)]. Colocalization was determined by ‘Just Another Colocalisation Plugin’ of ImageJ software, as described [9 (link),19 (link)].

COS7 cells were transfected with the VSV-G expression plasmid together with the EZ-TA or EZ-TD expression plasmid in slide chambers. The transfected cells were permeabilized with methanol 2 days after the transfection, and treated with mouse anti-VSV-G and goat anti-ezrin antibodies and then with Cy3-conjugated anti-mouse IgG (Sigma-Aldrich) and FITC-conjugated anti-goat IgG antibodies. The cells were observed under a confocal fluorescent microscopy (Olympus).

Cells were allowed to adhere to the coverslips were fixed with cold methanol (−20°C), rehydrated with phosphate-buffered saline (PBS) and then blocked in 3% bovine serum albumin in PBS. Immunostaining was performed by incubating the fixed cells with the primary antibody for 1 h at room temperature. The following primary antibodies were used: fluorescein isothiocyanate (FITC)-conjugated anti-HA monoclonal antibody (1:400 dilution; Sigma-Aldrich), L8C4 (anti-PFR2 monoclonal antibody [MAb]; 1:50 dilution) (31 (link)), anti-TbSAS-6 polyclonal antibody (1:400 dilution) (11 (link)), anti-TbBLD10 polyclonal antibody (1:400 dilution), and YL 1/2 (1:1,000 dilution; Millipore). The following secondary antibodies were used: Cy3-conjugated anti-mouse IgG (1:400 dilution; Sigma-Aldrich), FITC-conjugated anti-rat IgG (1:400 dilution; Sigma-Aldrich), and Cy3-conjugated anti-rabbit IgG (1:400 dilution; Sigma-Aldrich). Cells were washed with PBS, mounted with Vectashield mounting medium containing 4′,6-diamidino-2-phenylindole (Vector Labs), and imaged with an inverted fluorescence microscope (Olympus IX71) equipped with a cooled charge-coupled-device (CCD) camera (model Orca-ER; Hamamatsu) and a PlanApo N 60×, 1.42-numeric aperture differential inference contrast objective. Images were acquired using the Slidebook software (version 5; Intelligent Imaging Innovations).

HEK293 cells were grown on glass coverslips (350,000 cells/35 mm diameter dish), transfected for 24 h, and fixed using 4% paraformaldehyde in phosphate-buffered saline (PBS) for 30 min at room temperature.
Cell cultures overexpressing only fluorescent fusion proteins were directly mounted with SlowFade reagent (Molecular Probes, Thermo Fisher Scientific, USA). Cell cultures overexpressing HA-DLG1 were permeabilized with TritonX-100 0.1% in PBS after fixation, and incubated overnight with anti-HA (12CA5 clone, Sigma Aldrich, St. Louis, MO, USA). Cy3 conjugated anti-mouse IgG, was used as secondary antibody (Chemicon International, Temecula, CA, USA).
In all fluorescent microscopy experiments, despite the different spectral properties of the tags eventually used for protein detection, E6 expression was shown in green, E7 expression in blue and DLG1 expression in red. The Pearson correlation coefficient (PCC), used as co-localization parameter, was performed employing the Coloc2 plugin from FIJI software [27 (link)].
Fluorescence microscopy images were collected with a Carl Zeiss LSM880 confocal microscope following the sequential acquisition mode (Carl Zeiss, Germany). A 63x NA 1.4 plan apochromat oil immersion objective was employed.
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HEK293 cells were grown on glass coverslips (250,000 cells/35 mm diameter dish), transfected or not, and after 24 h fixed using 3.7% paraformaldehyde in phosphate-buffered saline (PBS) for 15 min at room temperature. Jurkat cells (10⁶) were seeded onto polylysine coated slides and fixed as described above. In both cases, after two washes with PBS, the cells were permeabilized with TritonX-100 0.1% in PBS, washed again, and incubated overnight with primary antibodies. Endogenous DLG1, GM130, and γ-Tubulin were visualized using anti-DLG1 (2D11 clone, Santa Cruz Biotechnologies, USA), anti-GM130 (EP892Y, Abcam, UK), and anti-γ-Tubulin (GTU-88, Sigma Aldrich, USA), respectively. HA-DLG1 was detected using an anti-HA (12CA5 clone, Sigma Aldrich, USA). Secondary antibodies used were Alexa 488-conjugated goat anti-rabbit IgG (Molecular Probes, Grand Island, NY, USA) and Cy3 conjugated anti-mouse IgG (Chemicon International, Temecula, CA, USA).