Zen 2012 sp1

Where indicated, images were subjected to adjustment of tonal range on the whole image using Adobe Photoshop (levels adjustment) to improve visualization for representation purposes. Such adjustments were identical for all images in a single assay. For quantification, raw images were analyzed. To assess Tfn uptake and CT-B binding, fluorescence intensity per cell was quantified using the ImageJ ‘measure’ tool. Individual cells were marked using the ‘selection tool’ and the integrated density value of the fluorescence signal in each cell was recorded. Cells with accumulation of Tfn in ERC-like structures were scored as described previously [21 (link)]. Analysis of Golgi morphology was performed as described earlier [22 (link)]. Colocalization analysis was performed in ZEN 2012 SP1 software (Zeiss). Briefly, a fixed threshold was set across all images to exclude background fluorescence and the percentage of colocalization of EEA1 with Tfn in the entire cell was calculated by the ratio of the number of colocalized pixels of EEA1 to the total EEA1 pixels.

A total of 2*10⁵ KATO III cells were seeded on cover slips in 12 well plate and incubated overnight for attachment. After that, the cells were washed three times with binding buffer and then incubated with 200 nM of Ep1 aptamer for 45 min at 4°C. Then, the cells were washed again for another three times with binding buffer and then fixed with 4% paraformaldehyde for 10 min at room temperature followed by washing three times with PBS. Cells were then transferred into glass slides and dibbed in mounting medium containing DAPI. LSM (laser scanning microscopy) images were captured with a confocal microscope (LSM 780, Zeiss) using a Plan Apochromat 20x/0.8 M27 objective, ZEN 2012 SP1 software (black edition, Zeiss) and the following settings: frame size 512 x 512; Pinhole Size 30 µm; laser 633nm; Master gain 780.

Following treatment, cells were fixed with 4% paraformaldehyde (PFA; 15 min), quenched with 0.1 M glycine-PBS (5 min) and permeablised using 0.1% Triton-X-100 (5 min). Cells were then incubated with the following primary antibodies: anti-Calreticulin (Cell Signalling, UK; #12238), anti-YAP1 (Abcam, UK; ab56701 or ab52771), anti-8OHdG (Abcam, UK; ab62623), anti-Hoechst 33342 (NucBlue^® fixed cell stain ready probes™, Life Technologies, UK; R37606). Secondary antibodies Alexa fluor^® 488 goat anti-mouse IgG (H + L) and Alexa fluor® 488 goat anti-rabbit IgG (H + L) (Thermo Fisher Scientific, UK; A-11001 and A-11034 respectively) and DyLight 649 horse anti-mouse IgG (H + L) and DyLight 649 horse anti-rabbit IgG (H + L) (Vector Laboratories, UK; DI-2649 and DI-1649 respectively) were used. Cells were imaged on a ZEISS LSM 780 confocal microscope (ZEISS Microscopy, UK) using the Plan-Apochromat 100x/1.4 Oil DIC objective (1024 × 1024 pixels; 8-bit. Pinhole 100 µm). Pseudo-colouring was applied to the images using Zen 2012 SP1 software (ZEISS; black edition, version 8.1): UV channel (blue), 488 channel (green) and 633 channel (red).

Immunostaining was performed on paraffin sections (for α-tubulin), cryosections or whole embryos fixed in 4% PFA or 10% trichloroacetic acid at 4 °C. Samples were incubated overnight at 4 °C with primary antibodies: Cldn1, −4 and −8 (Invitrogen, Carlsbad, USA, 1:25–1:50), Cldn3 (Abcam, Cambridge, UK, 1:50), Cldn14 (Sigma Aldrich, Oakville, Canada, 1:25), ZO-1 and ZO-2 (Invitrogen, 1:50, Carlsbad, USA), anti-disphospho-myosin light chain (Thr18/Ser19) (Cell Signaling, Ipswich, USA, 1:50), RhoA and Cdc42 (Santa Cruz, Santa Cruz, USA, 1:50), Par3 (Millipore, Etobicoke, Canada, 1:250), Dlg1 (US Biologicals, Salem, USA, 1:50), E-cadherin (BD Transduction, San Jose, USA, 1:100), α-tubulin (Abcam, Cambridge, UK, 1:100) or Vangl2 (gift from Dr. M Montcouquiol, 1:500). Alexa Fluor-conjugated secondary antibodies (1:500) were added for 1 h at RT. F-actin was detected using Alexa Fluor-conjugated Phalloidin (Molecular Probes, Eugene, USA). Sections and flat-mounted embryos were coverslipped with SlowFade Gold with DAPI (Molecular Probes, Eugene, USA) and imaged using a Zeiss LSM780 laser scanning confocal microscope. Colocalization and immunoquantification was performed using ZEN 2012 SP1 software (Carl Zeiss Microscopy, Germany).

The structure and morphology of the powders were measured using a LSM 710 Carl Zeiss Confocal Laser scanning microscope (Carl Zeiss, Oberkohen, Germany), by using four types of lasers, such as a diode laser (405 nm), Ar-laser (458, 488, 514 nm), DPSS laser (diode pumped solid state—561 nm), and HeNe laser (633 nm). The images were captured and rendered with the black edition of the ZEN 2012 SP1 software (Carl Zeiss, Oberkohen, Germany). The powders’ fluorescence was assessed both in their unlabeled (native) and labeled with the Red Congo (40 μM) fluorophore.

The structure and morphology of the microencapsulated powder obtained by co-microencapsulating the biologically active compounds from cornelian cherries and L. casei 431^® freeze-dried culture within the WPI, chitosan and inulin biopolymer matrix were observed and determined with LSM 710 Carl Zeiss Confocal Laser scanning microscope (Carl Zeiss, Oberkohen, Germany). The aforementioned system uses four types of laser, namely a diode laser (405 nm), Ar-laser (458, 488, 514 nm), DPSS laser (diode pumped solid state–561 nm) and HeNe laser (633 nm). The images were captured and rendered with the black edition of the ZEN 2012 SP1 software (Carl Zeiss, Oberkohen, Germany). The microencapsulated samples’ fluorescence was assessed both in their unlabeled (native) and labeled with the Red Congo (40 μM) fluorophore.