Wga alexa488

Briefly, the cardiomyocytes or deparaffinized heart sections (5 µm intervals) were subsequently fixed with 4% paraformaldehyde for 20 min and permeabilized with 0.5% (v/v) Triton X-100 in PBS for 20 min at 37 °C. After wash, the samples were blocked in 2% (w/v) BSA in PBS for 2 h and incubated with primary antibody at 4 °C overnight. In our studies, the following primary antibodies were used: troponin T-FITC (Abcam, ab196384, the cardiomyocytes marker) and WGA-Alexa488 (Thermo Fisher Scientific, W11261). After incubation and washing with PBS, the nuclei were stained with DAPI for 15 min. Images were captured with a confocal laser scanning microscope (Leica TCS SP8, Wetzlar, Germany). The total tissue positive areas were measured with ImageJ software (version 1.8.0). Data were expressed as a percentage of positive staining area to the total area.

After the completion of the experiments, the animals were euthanized. The hearts were harvested and arrested in diastole with 0.5% KCl (w/v) in PBS. The heart weights (HW) were determined and normalized to the body weight (BW). Then, part of each heart was immersed in 4% paraformaldehyde perfusion at 4 °C overnight. On the next day, the tissue samples were transferred to 70–100% ethanol for dehydration before embedding in paraffin. Heart sections (5 µm intervals) were deparaffinized and stained with hematoxylin and eosin (H&E), picrosirius red staining (Sigma-Aldrich) according to routine procedures. Images were acquired using Nikon Eclipse Ni light microscopy.
To determine the myocyte cross-sectional area, heart sections were incubated with wheat germ agglutinin-Alexa488 (WGA-Alexa488, Thermo Fisher Scientific, W11261) for 1 h to visualize the membranes and with 4,6 diamidino-2-phenylindole (DAPI) for 20 min to observe the nuclei. At least 100 randomly selected myocytes were measured from five animals/groups, and the average values were used for analysis. All measurements were made using ImagePro Plus software version 7.0 (Media Cybernetics, Rockville, MD). All morphometric analyses were performed in a blinded fashion. Sections were measured using a Leica TCS SP8 Confocal microscope (Leica, Wetzlar, Germany). All histopathologic data were confirmed by a blinded pathologist.

Heart tissue was fixed in 4% paraformaldehyde solution for 24 h and embedded in paraffin after dehydration using 70%–100% ethanol. The heart paraffin blocks were cut transversely into 5 μm sections. After deparaffinization, the heart sections subjected to Masson's trichrome staining were performed according to instruction book. To determined myocyte cross‐sectional area, heart sections were stained with Alexa Fluor 488 conjugated wheat germ agglutinin (WGA) (WGA‐Alexa488, Thermo Fisher Scientific, W11261) for 1 h to demarcate the cell boundaries. After staining, images were acquired with a confocal laser scanning microscope (Leica TCS SP8, Wetzlar, Germany). The myocyte cross‐sectional area and LV collagen volume were quantitatively measured by ImagePro Plus software version 7.0 (Media Cybernetics, Rockville, MD).

Intestinal mucus was visualized on-chip using wheat germ agglutinin (WGA)-Alexa 488 (ThermoFisher).⁵ (link) Briefly, 25 μg/mL of WGA in Hanks’ Balanced Salt Solution (HBSS, Gibco) was flowed at 200 μL/h through the apical channel for 30 min and then washed with continuous flow of HBSS at the same flow rate for 30 min. The entire channel was visualized with a fluorescence microscope (Excitation/Emission = 488/523 nm, Zeiss Axio Observer Z1). The mucus layer was set as the distance between the apical cell surface and the upper limit of the WGA-labeled material; the thickness of the mucus layer was measured by ImageJ. For MUC2 visualization, immunofluorescence microscopic imaging was carried out on 150–200 μm chip sections using antibodies against MUC2 (Santa Cruz, sc-15334) and a secondary antibody (ThermoFisher, A31573).¹² (link)^,13

Cellular uptake of TH-NPs was assayed in HaCaT cells seeded in an 8-well µ-slide (Ibidi®) following the same methodology as described before. Cells were incubated with or without R-TH-NP for 2 h at the indicated concentration, using FBS/phenol red free medium. Cell membranes were stained with wheat germ agglutinin (WGA) Alexa-488 (Molecular Probes) at 1 µg/mL for 15 min followed by fixation with paraformaldehyde 3% for 25 min. Cell nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI, Sigma Aldrich, Spain) at 0.5 µg/mL for 15 min. Internalization of NPs in HaCaT cells was assessed by confocal microscopy (Leica TCS SPII), 63× oil immersion objective lens [43 (link)]. Images were processed using Fiji image software.

The proximal and distal areas of the colon were dissected out and fixed in 10% PFA. OCT-embedded sections of 20 μm were stained overnight (O.N) at 4°C with mouse anti-E-Cadherin (Santa Cruz ref. sc-21791, 1:200) for immunohistochemistry characterization. After washing with PBS 0.01 M, the secondary antibody donkey anti-mouse Cy5 (Jackson Immunoresearch Laboratories, 1:400) was incubated for 2 h at room temperature. For lectin immunohistochemistry, WGA-Alexa488 1 μg/mL (Molecular Probes, Waltham, MA, United States) and DAPI 1 μg/mL (Molecular Probes, MA, United States) were incubated for 20 min before rinsed and mounted with fluorescence medium (Sigma). A tilt scan was done in an LSM780 (Zeiss) microscope, and the results of the total area (μm²) of mucin granules were quantified using the Otsu threshold (Otsu, 1979 (link)) of ImageJ since it is an option with excellent performance at different size of particles and in multi-intensity. Then, the results were normalized by the total mucosa layer (manually selected to compensate for the variation in the cross-sectional area of the colon).