Alexa fluor 488 labeled goat anti mouse secondary antibody

Fetal bovine serum (FBS), Dulbecco’s
modified Eagle’s medium (DMEM), Trypsin-EDTA, CCK-8, DBCO-Cy3,
Triton X-100, TGFβ1, 4′,6-diamidino-2-phenylindole (DAPI),
and primary antibodies against human alpha smooth muscle actin (α-SMA,
clone 1A4) and produced in mice were purchased from Sigma (St. Louis,
MO, USA). Primary rabbit anti-human collagen type 1 (COL-1) was bought
from Cedarlane Laboratories (Burlington, Canada). Phalloidin Alexa
Fluor 568, Calcein Am, and TOTO-3 were purchased from Invitrogen.
Alexa Fluor 488 labeled goat anti-mouse secondary antibody was obtained
from Molecular Probes Life Technologies. Alexa Fluor 488 labeled goat
anti-rabbit secondary antibody was from Invitrogen (Thermo Fisher
Scientific, United Kingdom). CNA35-OG488 was obtained from the Department
of Biomedical Engineering (TU/e Eindhoven, The Netherlands). For purified
water, Milli-Q was used.

For immunofluorescence staining, the 3-D cultured cells were fixed in formalin for 30 minutes at room temperature followed by permeabilization with 0.5% Triton X-100, then two blocking steps were used for 3-D immunofluorescence staining. After blocking in staining buffer (130 mM NaCl, 7mM Na₂HPO₄, 3.5 mM NaH₂PO₄, 7.7 mM NaN₃, o.1% BSA, 0.2% Triton X-100 and 0.05% Tween-20) supplemented by 10% normal goat serum for 1 hour at room temperature, the blocking buffer and goat anti-mouse F(ab’)2 fragment (Jachson ImmunoResearch #115–006–006) were added to cells on slides. Samples were incubated with primary antibodies. The Alexa Fluor 488-labeled goat anti-mouse secondary antibody was used (Molecular Probes, Eugene, OR) and the cells were counterstained with DAPI. Slides were mounted with fluorescent mounting medium (DAKO) and examined with fluorescence microscope. 50–100 cells were analyzed and the foci were counted by two researchers.

Aniline, ammonium persulfate (APS), sodium
diphenylamine-4-sulfonate, hydrogen chloride (HCl), Dulbecco’s
modified Eagle’s medium (DMEM), fetal bovine serum (FBS), trypsin-ethylenediaminetetraacetic
acid (EDTA), CCK-8, Triton X-100, transforming growth factor beta
1 (TGFβ1), 4′,6-diamidino-2-phenylindole (DAPI), primary
antibodies against human alpha-smooth muscle actin (α-SMA),
and myosin heavy chain (MyHC) were obtained from Sigma-Aldrich (St.
Louis, MO, USA). Phalloidin Alexa Fluor 568, Calcein-AM, and TOTO-3
were purchased from Invitrogen (Thermo Fisher Scientific, United Kingdom).
CNA35-OG488 was obtained from the Department of Biomedical Engineering
(TU/e Eindhoven, The Netherlands), and Alexa Fluor 488 labeled goat
antimouse secondary antibody was purchased from Molecular Probes Life
Technologies.

This study conducted IF staining according to previous report [16 (link)]. ADAM9antibody (1:25, Santa Cruz, USA) was dissolved with 1% bovine serum albumin (BSA). The Alexa Fluor 488-labeled goat anti-mouse secondary antibody (Molecular Probes, Eugene, OR) was also adopted for incubation. The images were captured under high-power magnification (х200) by confocal microscopy (Leica Microsystems Imaging Solutions, Cambridge, UK) with identical setting parameters.

The formation of multinucleated syncytiotrophoblast was assessed as we have described previously [21 (link)–24 (link)]. Trophoblasts were cultured in 2-chamber glass LabTek slides (Thermo-Scientific, USA). Specific treatments and incubation times are given in Results. The cells were then fixed and permeabilized using 3.7% formaldehyde/0.2% triton X-100 after which they were incubated in PBS/gelatin. Cell-cell junctions and nuclei were revealed by staining cultures simultaneously with a mouse monoclonal anti-E-Cadherin antibody (Abcam, Inc., USA) and 4',6-diamidino-2-phenylindole (DAPI) [25 (link)], respectively. The primary anti-E-Cadherin antibody was detected using AlexaFluor-488-labeled goat anti-mouse secondary antibody (Invitrogen, USA). The slides were mounted using glycerol vinyl alcohol (GVA) aqueous mounting medium (Zymed, USA) and examined using an epifluorescence microscope. Images were captured using an Optronics DEI750 CCD camera and Q imaging software. Identical exposure settings were used for all captured images. Total numbers of nuclei (T) from three random fields per well were counted (200–400 nuclei per field of view). The total number (S) of syncytia (cells containing three or more nuclei) and the total number of nuclei in syncytia (N) were also counted. A Fusion Index (FI) was calculated using the formula (N-S/T)x100.

1 × 10⁶ LS174T cells were detached with trypsin, centrifuged and incubated with an EGFR-specific antibody (1:100; monoclonal mouse IgG1 - Dako, Glostrup, Denmark) or with an IgG-anti-mouse antibody (BD Bioscience, Franklin Lakes, USA) as negative control in FACS buffer (PBS with 10% FBS) for 1 h on ice. Cells were washed with FACS buffer and incubated with an AlexaFluor 488 labeled goat anti-mouse secondary antibody (1:400 - Invitrogen, Langenselbold, Germany) for 1 h on ice. After washing, cells were resuspended in FACS buffer and flow cytometry analysis was performed on a BD Accuri C6 flow cytometer (BD Bioscience, Franklin Lakes, USA). Cells were gated by forward/sideward scatter and pulse width for exclusion of doublets. PI (propidium iodide, Sigma-Aldrich) was used for discrimination between viable and dead cells.

To measure protein synthesis in single living cells, we adapted a protocol to detect the incorporation of puromycin by fluorescent staining (51 (link)). Forty-eight hours post-transient transfection of wild-type or mutant RACK1-HaloTag in HEK293 cells, we replated the cells. We treated the cells with either the p38 mitogen-activated protein kinase (MAPK) inhibitor SB203580 (Sigma-Aldrich) at 10 μM or dimethyl sulfoxide (DMSO) for 8 h. Then, 30 min prior to fixation, we added the HaloTag TMR ligand (Promega) to the medium, followed by supplementation with 9.2 μM puromycin (Gibco) 20 min prior to fixation. We changed the medium 10 min prior to fixation in order to remove any excess HaloTag TMR dye. We incubated fixed samples with an antipuromycin antibody (clone 12D10; Millipore) for 1 h at room temperature and then with an Alexa Fluor 488-labeled goat anti-mouse secondary antibody (Invitrogen) according to the manufacturer's instructions.
We quantified fluorescence in the cells with a FACSAria III system (BD Biosciences) and selected and analyzed populations with FlowJo, v10.3 (Tree Star).