Alexa 546 secondary antibody

In vitro and in vivo constructs were fixed with 1% (w/v) PFA for 2 h, followed by 25% (w/v) sucrose incubation for 12 h. Samples were then embedded in optimal cutting temperature (OCT) compound (CellPath, Newton, UK), frozen in freezing isopentane, and cryosectioned (12 μm thickness). Cryosections were incubated for 1 h in 0.3% w/v Triton X-100 and 2% v/v normal goat serum, or 5% v/v donkey serum in PBS (blocking buffer), and then for 1 h in the following primary antibodies: mouse anti-PECAM-1 (CD31, endothelial cells, 1:100, LubioScience Gmbh, Zürich, Switzerland), rabbit anti-Ki67 (proliferating cells, 1:100, Abcam, Cambridge, UK), rabbit anti-phospho-histone H3 (Ser28) (1:400, Cell Signaling Technology, Danvers, MA, USA) and rabbit anti-cleaved caspase3 (cells in apoptosis, 1:100, Cell Signaling, Technology Inc., Danvers, MA, USA), mouse monoclonal anti-Human Nuclei (HuNu) (clone 235-1, human cells, 1:100, Merck Millipore, Burlington, MA, USA), and goat anti-Ve-Cadherin (endothelial cells, 1:200, Santa Cruz Biotechnology, Dallas, TX, USA). Tissue sections were then incubated in the dark for 1 h in fluorescently labeled Alexa488 and Alexa546 secondary antibody (dilution 1:200, Thermo Fisher Scientific Inc., Waltham, MA, USA). Cell nuclei were stained with 4′,6-Diamidine-2′-phenylindole dihydrochloride (DAPI, 300nM, Thermo Fisher Scientific Inc., Waltham, MA, USA).

To determine whether the nanoemulsions could interfere with cellular adhesion, we performed a cell adhesion test by immunocytochemistry, as described for morphological analysis. Briefly, the CESC, EuESC, and EctESC cultures in equal number (1 × 10⁴ cells/well) were treated for 24 h, fixed, permeabilized, and incubated with anti-β-catenin primary antibody (1:100, Sigma-Aldrich) followed by Alexa 546 secondary antibody (1:300, Invitrogen). Then, the cells were incubated with DAPI, the coverslips mounted onto histological slides and photographed in a fluorescence microscope (Nikon, Tokyo, Japan). The images shown are representative of at least three separate experiments.

Animals were sacrificed by CO₂ asphyxiation, tumors and organs were excised, cut in half and embedded in OCT, and immediately placed on dry ice. Samples were stored at −80 °C. Ten, 10 μm tumor cryosections were cut using a Cyrostar HM560 (Microm International, Waldorf, Germany), air-dried, and imaged for exogenous marker native fluorescence, mKate (visualized at 633 nm). Sections were fixed in 50% (v/v) acetone/methanol for 10 min at room temperature and subsequently stained for: (1) CD31 using a rat monoclonal anti-mouse CD31 antibody (BD Pharmingen) and Alexa 647 secondary antibody (Invitrogen), (2) Her2/neu using Herceptin, a monoclonal anti-human Her2/neu antibody (Genentech/Roche), followed by an Alexa 546 secondary antibody (Invitrogen), and (3) cell nuclei using Hoechst 33342, Bis-Benzimide (Sigma) (8 μg/mL at 37 °C), for 30 min.

For cFOS analysis, mice were treated s.c. with a single dose of 150 nmol kg⁻¹. Tissues were harvested 90 min following drug exposure. Fixed brains were coronally cryosectioned and 35-μm-thick slices were immunolabelled with the monoclonal rabbit anti-cFOS antibody (1:400, Invitrogen, no. MA5-15055) and the antirabbit Alexa546 secondary antibody (1:4,000, Invitrogen, no. A10040). According to the Allen mouse brain atlas, the ARC and the dorsomedial and ventromedial hypothalamic nuclei were captured at ×20 magnification using the Leica SP8 confocal microscope. In each region, the number of cFOS positive cells was counted in a blinded manner using Fiji/ImageJ software. For assessment of central nervous system drug appearance, mice (n = 3–4 each group) were treated with a single s.c. dose of the Cy5-labelled peptides (150 nmol kg⁻¹). After 90 min, tissues were harvested and processed similar to the cFOS analysis.

Cells were fixed with 4% paraformaldehyde and a standard immunocytochemistry procedure was performed. Visualization of the primary anti-HDAC3 antibody (Imgenex, San Diego, CA, USA). was done with the Alexa 546 secondary antibodies (Life Technologies). Nucleus was visualized by Hoechst 33258 (Sigma Aldrich). Images were obtained using an Axioplan 2 fluorescent microscope and Axiocam HRc CCD camera system (Zeiss, Göttingen, Germany).