Observer z1 inverted microscope

Transfected U2OS cells were plated with poly-L-lysine (Cultrex, cat.# 3438-100-01) coated glass coverslips. Cells were plated at a density of 150 000 cells per well in a 6-well plate and allowed to rest over night. Cells were fixed with 3% paraformaldehyde in PBS for 10–15 min, permeabilized with PBS with 0.5% Triton-X-100 for 10 min and blocked with 5% BSA in PBS for 15 min at room temperature. Primary and secondary antibodies were diluted in 1% BSA in PBS and incubated at 37°C for 1 h. Mounting of the coverslips onto microscope slides was done with Vectashield Hardset Mounting Medium with 4-,6-diamidino-2-phenylindole (DAPI) (Vector Labs, Burlingame, CA, USA). Coverslips were visualized and imaged using a Zeiss Observer.Z1 inverted microscope and AxioCam camera with Axiovision software (Carl Zeiss Microscopy GmbH, Jena, Germany).

Bright field images were taken on an Olympus IX51 inverted microscope at 4x, 10x and 20x magnification. Fluorescent images were taken on a Zeiss Observer Z1 inverted microscope at 20x magnification.

UROtsa cells or T24 cells were seeded on glass coverslips until they formed a confluent layer. Cells were treated with DiO-loaded Chi-NCs (2.8 × 10¹² particles mL⁻¹), in RPMI-1640 without FCS for 2 or 24 h. After two washing steps with HEPES buffered Ringer’s solution, the cells were fixed with 4% paraformaldehyde (Electron Microscopy Sciences). Cell surfaces were stained with Texas-red-conjugated WGA (Thermo Fisher) diluted in HEPES buffered Ringer’s solution (1:1000); nuclei were stained with 4′,6-diamidino-2-phenylindole. After three further washing steps, coverslips were embedded into a 1,4-Diazabicyclo-(2,2,2)octan/Mowiol (Sigma Aldrich) and mounted on a glass object slide. Samples were imaged using an Observer z1 inverted microscope equipped with a structured illumination module (ApoTome, Zeiss). Images were analysed with Zen software (version 1.1.2.0, Zeiss).

Fluorescent microscopy was performed on a Zeiss Observer Z1 inverted microscope. Live embryos and larvae were anesthetized with tricaine and spotted onto glass slides in E3 media for imaging.
Confocal microscopy was performed on an Andor XD Revolution Spinning Disk upright microscope. Live embryos were anesthetized in 120 μg/ml tricaine and then mounted in 0.75% low melting point agarose on 35mm glass bottom microwell dishes with a 20mm microwell and No. 1.5 coverglass (MatTek Ashland, MA) which was then flooded with E3 media supplemented with PTU and 120 μg/ml tricaine.
Fluorescent images were processed and analyzed using Zen 2.5 imaging software (Zeiss). In order to quantify infection burden images were taken using a 2.5x objective and the area and mean intensity of fluorescence were measured and used to calculate the log-Integrated Density of fluorescence which has previously been shown to correlate to infection burden [63 ]. Quantification of either Nile red or filipin staining in whole larvae was performed by imaging animals using a 2.5x objective and then measuring log-ID of fluorescence for either Nile red (TdTomato) or Filipin (DAPI) in the region of the animal from the anus to the end of the tail.

For E-cadherin immunostaining, embryos were dissected at E9.0 to E9.5 and were stained in whole mount with anti-E-Cadherin antibody (Life Technologies, 1:1000 dilution). A biotinylated anti-rat IgG secondary antibody (Vector Laboratories), followed by Vectastain Elite ABC (Vector Laboratories) and TSA fluorescein tyramide reagent (PerkinElmer) amplification, were used to detect anti-E-cadherin as described [36 ]. Embryos were photographed using a Zeiss Observer Z1 inverted microscope.
To compare E-cadherin staining intensity, mutant and control embryos were fixed in 4 % paraformaldehyde at 4 °C overnight, cut by vibratome, then stained with anti-E-Cadherin antibody (Life Technologies, 1:1000 dilution), using an Alexa-488-conjugated anti-rat IgG secondary antibody (Life Technologies, 1:1000 dilution) as described [37 ]. Images were collected using a Zeiss LSM510 laser scanning confocal microscope (data not shown).

High resolution imaging was performed using a Zeiss LSM780 system fitted on an Observer Z1 inverted microscope equipped with a LD LCI C-Apochromat 40x/1.1W objective (Zeiss). The 488 nm excitation wavelength of the Argon/2 laser, a main dichroic HFT 488 and a band-pass emission filter (BP490-535 nm) were used for selective detection of the green fluorochrome. The 543 nm and 633 nm excitation wavelength of the HeNe1 laser, a main dichroic HFT 488/543/633 and a long-pass emission filter (BP553-624 nm) (BP652-735 nm) were used for selective detection of the red fluorochrome and far-red fluorochrome. A 405 nm blue diode, a main dichroic HFT 405 and a band-pass emission filter (BP415-468 nm) were used for selective detection of the DNA counterstain. Single optical sections were acquired sequentially with a zoom factor of 1 and optimal (1 Airy unit) pinhole (scaling (x-y-z): 0.21 × 0.21 × 0.53 micron) and stored as 8-bit proprietary czi files. Single plane images were displayed using Zen2010 software (Zeiss) and exported as 8 bits uncompressed TIF images.

Digital images were captured on a Nikon TiE inverted microscope (Nikon Instruments), a Zeiss Observer.Z1 inverted microscope (Zeiss) or a Zeiss LSM 710 confocal microscope (Zeiss). The motorized stage and automated image-stitching parameters were used to obtain complete images of each tumour/tissue array. Immunohistochemical stains were captured using brightfield optics and a Nikon DSRi2 colour camera at ×20 magnification. Multi-channel immunofluorescent samples were captured on a CCD Hammamatsu Camera at ×10 magnification and an AxioCam MRm (Zeiss) at ×10 and ×20 magnification, using independently controlled excitation and emission wavelength filter-wheels for each specific fluorophore. NIS Elements software v.4.60.00 (Nikon) and Zeiss Zen 3.0 software (Zeiss) were used for image quantification and pseudo-colouring of immunofluorescent images where appropriate. Also, ImageJ Fiji^{81 (link)} was used for image quantification. Parameters in the tube formation assay were analysed in ImageJ Fiji using a code for the morphometric analysis of ‘Endothelial Tube Formation Assay’^{82 (link)}. Imaris 9.8.1 imaging software was used to analyse images capture by confocal/multiphoton microscope.