Fluoview 300 confocal microscope

In a subset of cells 0.2% biocytin was included in the intracellular solution to allow post-hoc morphological identification of GoCs. Slices were post fixed in 4% paraformaldehyde (PFA) for 24 hr. Free floating sections were washed with 1X PBS and rinsed with 0.3M glycine and 0.5% Triton-X 100. Slices were blocked in TBS containing 10% normal goat serum, 3% bovine serum albumin, 1% glycine, and 0.4% Triton-X 100 for 1 hr at room temperature. After the initial block, slices were incubated with Streptavidin conjugated Alexa 647 (1:1000; Invitrogen, Waltham, MA) overnight at 4°C. In cases of transgenic CRH-ires-CRE floxed ChR2-EYFP, slices were also incubated (overnight at 4°C) with rabbit anti-EGFP (1:1000; Invitrogen) to amplify staining of the EYFP-fused ChR2 protein. After staining, slices were rinsed with PBS and mounted with Vectashield anti-fade reagent (Vectorlabs, Burlingame, CA). Images of cells (Alexa 647) or CFs (ChR2-EYFP) were acquired using a 20X oil-immersion objective (0.85 NA) on an Olympus FluoView 300 confocal microscope using a 633 nm or 488 nm excitation wavelength. Images were processed using ImageJ software (NIH, Bethesda, MD).

Cells were fixed with 80% acetone for 10 min, permeabilized in 0.1% Triton X-100 in PBS for 10 min, and blocked in PBS containing 5% (v/v) normal goat serum for 30 min at room temperature. Samples were then incubated with primary antibody for overnight at 4 °C followed by staining with Alexa 488-conjugated goat anti-rabbit IgM (Invitrogen, Carlsbad, CA, USA), and counterstained with propidium iodide (PI) for 2 h. Samples were imaged by Olympus FluoView™ 300 confocal microscope. For transmission electron microscopy, rVvhA treated cells were fixed in Karnovsky’s solution and 2% OsO₄ in 0.1 M cacodylate at 4 °C for 2 hours, respectively. The cells were rinsed with distilled water briefly and stained with 0.5% uranyl acetate en bloc solution for overnight. After dehydration in graded ethanol series, samples were infiltrated with Spurr’s resin. The samples were imaged by JEM1010 (JEOL, Tokyo, Japan) transmission electron microscope operating at 80 kV.

IF experiments were essentially carried out as described earlier [45 (link)]. In brief, experimental cells were washed two times with ice cold PBS. Four percent paraformaldehyde containing 0.1% TritonX-100 were used to fix and permeabilize the cells for next steps. Again cells were washed with ice cold PBS and blocked using freshly made 5% skim milk for 1 hr. For LANA and Par3 co-localization experiments, we used Ramos, HEK-293, BJAB, BCBL1, BC-3, JSC-1, BC-3-shControl, BC-3-shLANA and 293BAC-KSHV cells. HEK-293 and 293BAC-KSHV cells were used to evaluate the localization pattern for Par3, E-cadherin, SNAIL, MMP9 and LANA. All primary antibodies as mentioned earlier were used for specific staining in cells. Evaluation of nuclear staining with different sets of proteins were carried out with DAPI (4,6-diamidino-2-phenylindole). After being washed with PBS, coverslips were mounted on glass slides using mounting medium. An Olympus Fluoview 300 confocal microscope was used in all experiments.

HEK293T or Saos-2 cells plated on coverslips were transfected with expression plasmids or not as indicated. Forty eight hours post-transfection, cells were fixed by 4% paraformaldehyde (PFA) including 0.1% Triton X-100 for 15–20 mins at room temperature [81 (link)]. B-cells were air-dried and fixed similar to above. The fixed cells were washed with 1×PBS for three times, and 5% Bovine serum albumin (BSA) was used for blocking. EBNA3C and Bcl6 were detected by mouse anti-EBNA3C (A10) and rabbit anti-Bcl6 antibody, respectively. The slides were examined using an Olympus Fluoview 300 confocal microscope, and Images were analyzed by Fluoview software (Olympus Inc., Melville, NY).

Visualization and analysis of the samples were carried out using an Olympus Fluoview 300 confocal microscope with an Olympus Fluoview FV1000S-IX81 image acquisition software system. Data were collected in four separate channels, including differential interference contrast (DIC), UV for CD4, virtual red for CD8, FITC for GR, red for Bak, Bax, Bcl-x_L, and Bim or red for mitochondria and FITC for Bax. Sequential scanning was used for image acquisition. Signals were collected from cells in 3–3 frames and Bak, Bax, Bcl-x_L, Bim-GR and CMX-Ros-Bax morphological association was analyzed with the ImageJ software (http://rsb.info.nih.gov/ij) using co-localization plug-in. Co-localization data was calculated using the original, unmodified images. Based on the analysis of pixel fluorescence intensities, ranging from 0 to 255, specific staining was distinguished from background by using a threshold value of 50 as described elsewhere [32 (link), 33 (link)]. Then, co-localized pixels between Cy3-GR and CMX-Ros-Bax were counted. One hundred DP cells per sample were analyzed altogether using this approach. Brightness and contrast of representative images have been adjusted.

The autolysosomes and lipid raft were immunostained with LysoTracker Red DND-99 (LysoTracker, Invitrogen Co., Carlsbad, CA, USA) and cholera toxin B subunit (CTB, Sigma-Aldrich), respectively. Immunocytochemical staining was performed as described in our previous report and counterstained with 300 nM DAPI in PBS for 5 min^{8 (link)}. The immunostained cells were visualized with an Olympus FluoView 300 confocal microscope with 400 × objective. The co-localization of protein with LysoTracekr or CTB was analyzed by using Metamorph software.

At the end of each experiment, the preparation was fixed overnight in 4% paraformaldehyde in phosphate-buffered saline (PBS). Preparations were then rinsed with PBS four times for 10 min each, pinned out dorsal side up, and cleared in an ascending ethanol series to methyl salicylate. Cleared whole mounts were mounted in methyl salicylate in a Permanox dish with a coverslip base. To prevent movement during imaging, a small, thick slip of glass was placed on top of the nerve cord.
Preparations were examined as whole mounts oriented for frontal view, dorsal side up (Fig. 1E). The structure of each labeled neuron was captured as a stack of confocal images that extended from the most dorsal to the most ventral part of the cell. Images were captured with an Olympus FluoView 300 confocal microscope (Olympus America, Center Valley, PA) equipped with krypton (488 nm) and argon (568 nm) lasers and an Olympus ×20 0.7 NA UPlanApo lens. Step size was 0.75 μm. The images were converted to 24-bit TIF images in FluoView software, where the gamma and intensity were adjusted to optimize the background intensity. The resulting images were then transferred to Adobe Photoshop for further adjustment of brightness, contrast, and sharpness. All images in each stack were adjusted uniformly.