Fitc conjugated secondary antibody

Three archival SCLC paraffin blocks collected from patients who died from their cancers with distant metastases were randomly chosen. Recut slides were first deparaffinized and treated with an antigen-retrieval reagent. These slides were immunostained using either non-immunized IgGs from different species, the mouse monoclonal anti-tyrosinase-related protein (Trp)-75 (also called Trp-1), goat anti-melanoma antigen (Mage)-1 polyclonal antibody [Lab Vision/Neomarkers (Fremont, CA, USA), or rabbit anti-telomerase reverse transcriptase (hTert) antibody (Santa Cruz. Biotech, Santa Cruz, CA, USA)]. The slides were washed and stained with the appropriate FITC-conjugated secondary antibodies (Vector Labs, Burlingame, CA, USA). Representative fluorescence images were captured using a Nikon Eclipse E600 fluorescence microscope with RTKE Spot Camera and Spot software.

Cells grown on glass coverslips were fixed with either 70% ethanol or 4% paraformaldehyde, permeabilized with 0.25% Triton X-100 (only for paraformaldehyde fixation), blocked with 10% FBS, and incubated with primary antibodies at 4 °C overnight. Cells were then incubated with FITC-conjugated secondary antibodies (Vector Laboratories) for 1 h at room temperature. Nuclei were counterstained with 4′,6-diamino-2-phenylindole (DAPI). For 3D growth assays, cells were seeded onto 8-well chamber slides coated with reduced growth factor basement membrane matrix Geltrex (Thermo Fisher Scientific) and cultured for 48 h in DMEM/F12 with 10% FBS, followed by fixation with 4% paraformaldehyde/20% sucrose, and staining with phalloidin-TRITC (Sigma). Nuclei were counterstained with DAPI. Images were obtained with an Olympus DSU IX83 confocal microscope using CellSens Dimensions software.

Cells were plated at a density of 40,000 cells/cm² on 8-chambered Nunc Lab-Tek Chamber Slide System slides (Thermo Fisher; Cat# 177402). Cells were allowed to attach for 24 h. After various treatments, cells were fixed in cold methanol for 10 min on ice. Cells were then permeabilized with 0.25% Triton X-100 (VWR; Cat# VWRVM143) in PBS before incubating with primary antibody against TLE1 (Abcam; ab183742; 1:200 dilution). Following 1 h of primary antibody incubation, FITC-conjugated secondary antibody (Vector Laboratories; Cat# FI-1000) was applied for 1 h at room temperature. Slides were mounted using a medium containing DAPI (Sigma-Aldrich; Cat# F6057). Images were taken using an Olympus BX-51.

Frozen tissue sections were placed in 0.3% H₂O₂/methanol for 10 min, washed twice in PBS for 5 min, and incubated with 3% horse serum in PBS/0.3% Triton X-100 for 30 min at room temperature. After draining the solution from the tissue section, the tissue was incubated with mouse anti-CD34 (SC-7324, Santa Cruz Biotech, Santa Cruz, CA), mouse anti-Synaptopodin (Q01246M, Meridian Life Science, Inc, Memphis, TN), rabbit anti–smooth muscle actin (SMA) (ab5694, Abcam, Inc., Cambridge, MA), mouse anti-Nestin (MAB353, EMD Millipore, Billerica, MA), mouse anti-RECA (MCA970R, AbD Serotec, Raleigh, NC), mouse anti-Stro-1 (MAB1038, R&D Systems, Inc., Minneapolis, MN). After rinsing with PBS, the sections were incubated with FITC-conjugated secondary antibody (Vector Labs, Burlingame, CA) for 60 minutes in 3% horse serum. For tracking EdU-positive cells, tissue sections were incubated with Click-IT reaction cocktail containing Alexa594-azide (Invitrogen) for 30 minutes at room temperature, then washed with 1 ml of 3% BSA in PBS. Nuclear staining was performed with 4,6-diamidino-2-phenylindole (DAPI, Invitrogen). The negative control experiments were also performed where the secondary antibodies or primary antibodies were omitted to exclude the potential non-specific binding to tissue specimen.

HepG2 and Huh-7 cells transfected with P4HB siRNA, control siRNA, P4HB, or control vector were grown on 2-well chamber slides (Nalge Nunc International, USA) for 48 h. The cells were subsequently incubated with anti-E-cadherin or anti-vimentin antibody at 4°C overnight. After washing with PBS, the cells were incubated with a FITC-conjugated secondary antibody (Vector Laboratories Inc., USA) at room temperature for 1 h. The cells were then stained with 4’, 6-diamidino-2-phenylindole (DAPI) and examined under a Leica SP2 MP confocal microscope (Leica-Microsystems, Germany).

HepG2 cells were plated on coverslips and incubated for 6 h with fatty acids. Cells were fixed with 4% paraformaldehyde, permeabilized with 0.2% Triton X-100, and blocked with 0.2% BSA/10% horse serum in PBS. Coverslips were incubated with eEF1A-1 monoclonal antibody (Millipore), followed by FITC-conjugated secondary antibody (Vector Laboratories). For visualization of ER, cells were incubated with anti-calnexin monoclonal antibody (Cell Signaling Technology) followed by secondary antibody conjugated to Alexa Fluor 546 (Molecular Probes). To visualize lipid droplets, cells were stained with Oil Red O (0.3% Oil Red O in isopropanol and PBS). To visualize polymerized actin (F-actin), cells were stained with rhodamine phalloidin (Molecular Probes). All coverslips were affixed to glass slides with mounting medium containing DAPI (Molecular Probes). Images were generated by confocal laser scanning microscopy (Zeiss LSM 510 Meta Confocal Microscope, London Regional Cell and In Vitro Molecular Imaging Facility). Co-localization was quantified in Image J using Pearson’s correlation coefficient (Rr) where 0 indicates random distribution and 1.0 indicates complete positive correlation between fluorescent signals.