Lsm 710 confocal scanning microscope

Primary hippocampal neurons were transfected with Mitochondria-GFP (Invitrogen) at DIV14, and fixed in 10% buffered formalin at DIV 20–22. Then, samples were incubated with anti-Flag (1:500 dilution, Origene, Rockville, MD) overnight at 4 °C. Cells were washed and incubated with Alexa-568 antibody (1:200 dilution, Invitrogen, Molecular Probes, Carlsbad, CA) for 1 h at room temperature and mounted on glass slides. Images were taken with Zeiss LSM 710 confocal scanning microscope (Zeiss, OberKDchen, Germany) and processed using ZEN software (Carl Zeiss Microscopy GmbH, Jena, Germany).

Confocal imaging was performed on an LSM 710 confocal scanning microscope (Zeiss, Oberkochen, Germany), controlled by ZEN 2011 (Zeiss) software, equipped with a Plan-Apochromat 63× objective (NA 1.4, oil), 488, 561 and 633 nm laser lines, and continuous spectral detection. Images were acquired at 16-bit depth, 1024 × 1024 pixels, pixel size 0.132 µm, with 2× line averaging and a pixel dwell time of 6.3 µs, either as a single plane or as a Z-stack with 0.37 µm step size. In all channels, pinhole was set to 1 Airy unit. Emission light was collected sequentially, according to the emission spectra of the fluorophores used.
For the live-cell imaging, a temperature module was used and cells were placed in a heating insert (PeCon, Erbach Germany), which had been equilibrated to 37 °C. The medium used for imaging was Hibernate E medium containing 1% PS and 2% B27 Plus.

HaESCs cultured in Chamber Slides were fixed by 4% paraformaldehyde solution for 15 mins at room temperature. Blocking and permeabilization buffer (1% BSA, 0.5% Triton X-100 in PBS) was used to permeabilize the cells for 1 hour at room temperature. Cells were then incubated overnight with Oct-3/4 (sc-5279; Santa Cruz) and Nanog (sc-33760; Santa Cruz) antibodies at 4 °C, and then incubated with fluorescent conjugated secondary antibodies for 1 hour at room temperature. Nuclei were counterstained with DAPI. Images were captured with Zeiss LSM 710 Confocal Scanning Microscope.

Lungs were collected and fixed in 4% buffered formaldehyde in PBS pH 7.2–7.4 (Bio Lab, Jerusalem, Israel) for 2 weeks. Sections of 5 µm were prepared after paraffin embedding using an RM 2255 microtome (Leica, Nussloch, Germany). Antigen retrieval was performed by incubation in Target Retrieval Solution (S1700, DAKO, Carpinteria, CA, USA, 30 min, 95 °C). After blocking in 5% BSA in PBS, slides were incubated (overnight, 4 °C) with purified anti-CD31 (390, Biolegend, San Diego, CA, USA), proSPC (Millipore, Temecula, CA, USA), podoplanin (T1α, 8.1.1, Biolegend), VE-cadherin (ab33168), claudin 5 (ab15106), connexin 43 (ab117843), occludin (ab31721) or claudin 18 (ab203563) (Abcam, Cambridge, MA, USA). Alexa Fluor 594- or 488-coupled donkey anti-rabbit or Alexa Fluor 594-coupled goat anti-Armenian hamster antibodies were used for detection (Molecular probes®, Thermo Fisher Scientific, Carlsbad, CA, USA). For nuclear staining, slides were mounted with Prolong® Gold antifade reagent containing DAPI (Molecular probes®, Thermo Fisher Scientific, Carlsbad, CA, USA). Analysis was performed using an LSM 710 confocal scanning microscope (Zeiss, Jena, Germany) equipped with the following lasers: argon multiline (458/488/514 nm), diode 405 nm, DPSS 561 nm and helium-neon 633 nm.

Anti-ricin Ab was conjugated (1 mg) with the Alexa Fluor 594 protein labeling kit (Molecular Probes, Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer’s instructions.
For immunolocalization experiments, HEK293 cells or a single cell suspension (SCS) from mice lungs were seeded on #1 glass cover slips in 24-well dishes and exposed to ricin (100 ng/ml). Cells were fixed with 4% paraformaldehyde (PFA, Gadot, Israel) for 10 min at 4 °C, washed three times with PBS, and placed for 1 hour in a blocking solution (10% normal goat serum (NGS)) in PBS containing 0.05% Tween-20 (P5927, Sigma-Aldrich). Cells were incubated in a 1:500 dilution of rabbit anti-LRP1 Alexa Fluor 488 (Abcam, Cambridge, MA, USA) and anti-ricin Alexa Fluor 594 in an antibody cocktail solution (50% blocking solution/0.05% Tween-20/PBS) for 24 hours at 4 °C. Cover slips were processed as described previously^{77 (link)} and imaged in a sequential manner using an LSM 710 confocal scanning microscope (Zeiss, Jena, Germany) equipped with following lasers: argon multiline: 458/488/514 nm); diode: 405 nm; DPSS: 561 nm; and heliumneon: 633 nm. The percent of colocalization was quantified using Zen software (version 2.1, 2008; Zeiss). Image parameters: Scan mode-plane; Dimensions- X:1691 Y:1127 8-bit; Average-8; Pixel dewl-1.27 µs.