Alexa 568 conjugated secondary antibody

Cells were seeded on eight-well chamber slides (Millicell EZ slide, Millipore). Before antibody incubation, slides were put on ice for 20 min. Cells were incubated with anti-β2AR antibody (Santa Cruz sc-569) at a dilution of 1:100 in phosphate-buffered saline (PBS) for 3 h. Cells were then fixed with 4% paraformaldehyde (EMD Millipore), washed with PBS, and incubated in Superblock blocking buffer (ThermoFisher Scientific) for 1 h, before incubation with Alexa-568-conjugated secondary antibody (Invitrogen) at a dilution of 1:200 in PBS for 1 h in the dark. Cells were then permeabilized with 0.2% Tx-100 (Pierce) to allow for nuclear staining and coverslips mounted with Vectashield plus DAPI (4′,6-diamidino-2-phenylindole) mounting medium (Vector Labs). Stained cells were viewed on an Olympus Fluoview 1000.

Cells were grown on sterilized glass coverslips. The cells were then fixed with 4% paraformaldehyde. For immunostaining, cells were blocked with 3% BSA in PBS, stained with a 1:1000 dilution of the primary antibody in PBS, and stained with 1:1000 Alexa 488-conjugated secondary antibody (Invitrogen, Carlsbad, CA, USA) or Alexa 568-conjugated secondary antibody (Invitrogen, Carlsbad, CA, USA). Images were captured using a Carl Zeiss LSM710 confocal microscope (Carl Zeiss, ObERKochen, Germany). The Image J software was used to analyze cell images (https://imagej.nih.gov/ij, accessed on 2 June 2021).

To assess the localization of vWF and α-SMA in liver tissues, cryosections (7 µm thick) were fixed in 100% methanol (Merck) and incubated with blocking solution (Dako) for 1 h at RT followed by anti-vWF (1:200, Abcam) and anti-α-SMA (1:400, Dako) primary antibodies at 4 °C overnight. The sections were incubated with Alexa 488- and 568-conjugated secondary antibodies (1:400; Invitrogen) for 1 h at RT. To evaluate the localization and expression of α-SMA in rat HSCs, T-HSCs treated with WKYMVm were fixed with 100% methanol. The cells were incubated with blocking solution (Dako) at RT for 40 min and a rabbit anti-α-SMA (1:200) antibody at 4 °C overnight. Subsequently, the cells were incubated with an Alexa 568-conjugated secondary antibody (1:400, Invitrogen) at RT for 1 h and counterstained with DAPI. Images were taken with a confocal microscope (LSM 700) and analyzed with ImageJ software. All experiments were performed in triplicate.

To investigate the localization of vascular epithelial growth factor receptor 2 (VEGFR2), β-catenin, and CD31 in ovarian tissues, 6-μm-thick frozen ovarian sections were treated with Blocking Solution (DAKO, Glostrup, Productionsvej, Denmark) at room temperature for 60 min and treated with primary antibodies against each target protein at 4 °C overnight. The following antibodies were used: rabbit anti-nonphospho-β-catenin (4270 S, Cell Signaling) diluted 1:100; rabbit anti-VEGFR2 (9698 S, Cell Signaling) diluted 1:100; and goat anti-CD31 (SC-1506, Stan Cruz Biotechnology) diluted 1:100. On the following day, the tissues were washed with phosphate-buffered saline (PBS) and incubated with an Alexa 488-conjugated secondary antibody or an Alexa 568-conjugated secondary antibody (Invitrogen) at room temperature for 60 min. Then, the sections were stained with 4,6-diamidino-2-phenylindole (DAPI) for nuclear counterstaining. The slides were observed by fluorescence microscopy (Nikon, Tokyo, Minato, Japan). All parts of each slide were observed, and representative images were captured for presentation.

Internalization assays were performed as described6 (link), with minor modifications. Briefly, hippocampal neurons at 15–18 days DIV were incubated with antibodies against the N-terminus of GluA2 (2 μg/ml, mouse monoclonal, clone 6C4, Millipore) for 30–60 min at 18–20 °C. After brief washing, neurons were either incubated with control medium or medium containing NMDA (50 μM) for 15 min at 37 °C. Subsequently, they were fixed for 5 min at room temperature in paraformaldehyde (4%)/sucrose (4%) without permeabilization. Surface-remaining antibody-labeled receptors were visualized by means of a 1-h incubation with saturated Cy5-secondary antibody (15 μg/ml, Jackson ImmunoResearch). Neurons were permeabilized for 2 min with methanol (−20 °C), and internalized antibody-labeled GluA2 was detected by a 1-h incubation with Alexa 568-conjugated secondary antibody (1 μg/ml, Invitrogen). Simultaneously, infected GFP-fluorescent neurons were stained with antibodies against GFP (5 μg/ml, Invitrogen, rabbit polyclonal). Thus, the GFP fluorescence of infected neurons was enhanced by a 30 min incubation with Alexa 488-conjugated secondary antibodies (4 μg/ml, Invitrogen). After staining, the coverslips were mounted in Mowiol (Sigma-Aldrich, Barcelona, Spain).

HeLa cells were grown on glass coverslips at 70–80% of confluence and treated with L. crispatus BC5, S. agalactiae, E. faecalis, or B. subtilis cells (5 × 10⁷ CFU) for 1 h as described above, then washed three times with PBS and fixed in paraformaldehyde. Samples were stained with anti-human CD49e (i.e., anti-human α5 integrin subunit) primary antibody (1:500 in 1% BSA/PBS, BioLegend, San Diego, CA, United States) overnight at 4°C, followed by incubation with Alexa 568-conjugated secondary antibody (1:1000 in 1% BSA/PBS, Thermo Fisher Scientific) for 1 h at room temperature. As a specificity control for α5 integrin subunit signal, untreated Hela cells were also stained with IgG isotype (1:500 in 1% BSA/PBS, Sigma-Aldrich), followed by Alexa 568-conjugated secondary antibody. Specimens were embedded in Mowiol and analyzed in confocal microscopy as described above. Fluorescence intensity was quantified on at least eight randomly chosen microscopic fields by using Image J; results were expressed as average fluorescence (A.U.)/field ± SD.