Three-dimensional structured illumination microscopy of liver sinusoidal endothelial cell fenestrations.

Materials

Reagents included Type 1A Collagenase (Sigma Chemical, St. Louis, MO #C9891), RPMI (Gibco Invitrogen #11875-093), CellMask Orange (Invitrogen, # C10045 ), Prolong Gold (Invitrogen # P36930 ), eNOS antibodies (BD Biosciences, #610298), Alexa Fluor 488 Phalloidin (Invitrogen, #A12379), and Alexa Fluor 532 Phalloidin (Invitrogen, # A22282 ).

Cell Culture

Experiments were performed in adherence with the guidelines outlined in the National Institutes of Health “Guide for the Care and Use of Laboratory Animals” (Revised 1985) prepared by the National Academy of Sciences as described previously ( DeLeve et al., 2006 ). The experiments followed protocols approved by the Animal Care and Use Committee of the University of Southern California. Male Sprague Dawley rats (body wt 250–280 g) were anesthetized with pentobarbital and then treated with 200 μL porcine intestine heparin (1,000 U/ml). The liver was perfused for 10 min with calcium-free Geys buffered saline at 37°C at 10 ml/min, followed by perfusion in a recirculating fashion for 20 min with GBS containing 0.05% type 1a collagenase (Sigma Chemicals, St. Louis, MO). The livers were mechanically dissociated, pressed through polypropylene mesh, and centrifuged, and the digest was resuspended in 50 ml GBS. The resulting digest was used to isolate LSECs by gradient centrifugation and centrifugal elutriation. Purity was 98% as determined by positive staining for fluorescent acetylated low-density lipoprotein and a peroxidase stain to reveal contaminating Kupffer cells. Viability was 95% and yield averaged >100 million cells. Cells were plated on #1.5 coverslips and cultured overnight in serum-free media. The following day the LSECs were fixed with 4% fresh paraformaldehyde in PBS. Following fixation the cells were prepared for visualization using SIM.

Fluorescence studies

To visualize the filamentous actin within LSECs the fixed cells were permeabilized with 0.05% saponin in blocking solution and then incubated with Alexa Fluor 488 Phalloidin (Invitrogen, #A12379) or Alexa Fluor 532 Phalloidin (Invitrogen, # A22282 ) for 10 minutes at room temperature. For cell membrane visualization, 0.5 ug/ml of Cell Mask Orange (Invitrogen, # C10045 ) was applied to the fixed cells for 10 minutes at room temperature. Cells were mounted with Prolong Gold (Invitrogen, # P36930 ) prior to examination. To visualize eNOS distribution, fixed cells were permeabilized with 0.05% saponin in blocking solution for 1 hour prior to an overnight incubation (4°C) with rabbit polyclonal anti-eNOS (BD Biosciences, #610298) (1:50). The following day cells then were treated with goat anti-rabbit antibody conjugated to Alexa Fluor 532 (Invitrogen, #A11009) or goat anti-rabbit antibody conjugated to Alexa Fluor 488 (Invitrogen, #A11034) for 45 minutes at room temperature. Cells were mounted with Prolong Gold (Invitrogen, # P36930 ) and examined with SIM.

Structured illumination microscopy

The structured illumination microscope is based on a prototype of the commercial model Deltavision|OMX V2.0 (Applied Precision Inc, Issaquah, WA). It delivers 488 and 532 nm lasers with variable power to the environmentally isolated microscope system using a multimode optical fiber and fiber shaker. The laser light passes through a movable phase grating where the grating’s image plane is projected onto the sample using a collimation lens, a 100× 1.40 NA oil objective (UpanSApo, Olympus, Japan), and 1.514 index immersion oil. Fluorescence emission is collected by the same objective, split by channel, and filtered using a FF01-512/25-25 fluorescence filter (Semrock, Rochester, NY) for the 488 excitation channel or a FF01-593/40-25 (Semrock, Rochester, NY) fluorescence filter for the 532 excitation channel. Each channel was imaged by a dedicated Cascade II 512 EM-CCD camera (Photometrics, Tuscon AZ), which used 10–70 ms exposures, 1 pre-amp gain, and variable EM gain. Acquisition and all mechanics were controlled by the OMXN controller software (Applied Precision Inc, Issaquah, WA) while reconstructions were made with the OMX specific SoftWoRx v4.5.0 software package (Applied Precision Inc, Issaquah, WA). The reconstruction algorithms takes into account the specific optical transform functions for each channel, which are wavelength-dependent. Each channel required 5 exposures for a given angle (5 lateral translations of the interference pattern by 72 degrees), and 3 different angular grating positions (in steps of 60 degrees) for a total of 3×5=15 exposures per optical slice. As each channel uses a different EMCCD camera, there were slight differences in the observed grating angles between channels. To obtain 3D images, the sample is translated vertically in steps of 125 nm, resulting in 16 vertical slices for a 2 μm thick sample.

Image analysis

Image analysis was performed using Volocity (Perkin-Elmer) and ImageJ software ( http://rsb.info.nih.gov/ij/ ). 2. 6 Statistics All data are presented as mean ± standard deviation and comparisons of two groups performed using a Students t-test.

Materials

Reagents included Type 1A Collagenase (Sigma Chemical, St. Louis, MO #C9891), RPMI (Gibco Invitrogen #11875-093), CellMask Orange (Invitrogen, # C10045 ), Prolong Gold (Invitrogen # P36930 ), eNOS antibodies (BD Biosciences, #610298), Alexa Fluor 488 Phalloidin (Invitrogen, #A12379), and Alexa Fluor 532 Phalloidin (Invitrogen, # A22282 ).

Supplementary Material 01 02 03 04