Lsm 700 confocal microscope

Primary PB cells were treated with vehicle or 30 µM NOR for 2 h. Acridine orange (final concentration = 2 µg/ml) was added 20 min before the end of treatment. Photo-oxidation was induced by exposing cells to blue light, and changes in green intensity (eGFP) were captured using ZEISS LSM700 confocal microscope. To determine lysosome location, primary tumor cells were treated with vehicle control or 12.5 µM NOR for 2 h. Acridine orange solution was added as above and lysosomes and cytoplasm were captured with red (mCherry) and green (eGFP) filters using ZEISS LSM700 confocal microscope, respectively. Location of lysosomes relative to nucleus was assessed using ImageJ software. Lysosomes were considered as juxtanuclear when they were within 4 µM-range from the nucleus, and perinuclear when they were outside the 4 µM-range.

To visualize the tonoplast with the fluorescent probe FM4-64 (Molecular Probes Inc., Eugene, OR, U.S.A.), Nt-JAT2-GFP-expressing tobacco BY-2 cells were incubated with 8 µM FM4-64 for 24 h at 25°C, and monitored for red fluorescence. The green fluorescence of the expressed Nt-JAT2-GFP protein was visualized with a Zeiss LSM700 confocal microscope. The fluorescence of GFP and FM4-64 were detected by 488/490–555 nm and 555/640 excitation/emission filter sets, respectively.
Nt-JAT2 was subcellularly localized in yeast cells using an Nt-JAT2-GFP fusion construct. The cDNA fragments carrying the coding regions of Nt-JAT2 and GFP were separately amplified, fused by crossover PCR and subcloned into the yeast expression vector pDR196. The fluorescence of Nt-JAT2-GFP was visualized with a Zeiss LSM700 confocal microscope (Carl Zeiss, Oberkochen, Germany) and the subcellular location of this protein in yeast cells was determined.

Flies were injected in their thorax with 69nl of pHrodo red or Alexa 488 bacteria (Life Technologies Inc.) using a nanoject injector (Drummond). The fluorescence within the abdomen of the flies was then imaged at 45min, 3hrs, and 5hrs post-injection with a Leica MZFLIII fluorescent microscope and DFC300 FX camera and quantified using Image J 2.0.0-rc-30/1.49s (NIH).
For ex vivo imaging, flies were injected with 46nl of PBS at 45min, 3hrs and 5hrs after infection to release all hemocytes, and 10 flies were bled on a lysine-coated slide by mechanically scraping their hemocytes onto a drop of PBS. Once settled for 10min on the slide, hemocytes were quickly dried and mounted with AF1 mounting solution (Citifluor Ltd). Slides were automatically scanned using a Zeiss LSM 700 confocal microscope, and the number of plasmatocytes and average fluorescence signal per plasmatocyte quantified.
For immunostaining, flies were bled as described above and the hemocytes fixed in a solution of PBS, Tween 0.1%, PFA 4% for 30min. The samples were incubated in PBT with 1% normal goat serum and Crq [53 (link)] and GFP antibodies (Roche) at 1:500 overnight at 4°C. Samples were washed at RT three times for 5min in PBS, incubated with the appropriate secondary antibodies at 1:1000 in PBT for 2hrs at RT, and washed three additional times in PBT. Samples were imaged with a Zeiss LSM 700 confocal microscope.

The micrographic images of the longitudinal optic nerve sections were obtained using a Zeiss LSM700 confocal microscope under 40Xagnification with automatic stitching and analyzed using Image J in a blinded fashion. Three longitudinal Z-stack sections were analyzed per each optic nerve tissue section and each experimental group included 4–6 mice. In some cases, the optic nerve micrographs were obtained after manually stitching the image sections using Photoshop. To analyze the extent of the axon growth after optic nerve injury, the number of the CTB-labeled regenerating axons (N) that passed through the measuring point at a specific distance (d) from the lesion site was estimated using the following formula as described earlier [31 (link)]: σd = πr² × [average axon numbers per millimeter]/t, where r is half the cross-sectional width of the nerve at the counting site (d), the average number of axons per millimeter nerve width was from three nerve sections, and t is the section thickness (12 μm). The number of the regenerating axons extended from the lesion site was determined at a 100 μm interval. The longest axon length was determined by analyzing the micrographs of two longitudinal optic nerve sections per animal, taken under 40X magnification using a Zeiss LSM700 confocal microscope (Zeiss).

Colon sections (5μm) were deparaffinized, rehydrated through a series of washes in graded ethanol and water, followed by an antigen retrieval step (by boiling the sections in 10 mM sodium citrate buffer, pH 6.0 for 20 min). Sections were then incubated in blocking solution (5% bovine serum albumin (BSA) + 0.3% Triton X-100 in PBS) for 1 h, followed by incubation overnight at 4°C with primary antibodies [p-ERK1/2, p-AKT, p-p38 MAPK, and COX-2) (1:100 dilution)], Cell Signaling, USA, diluted in 1% blocking solution). On the following day, sections were washed and incubated with and the secondary antibody conjugated to Alexa Fluor 555 [Goat anti rabbit SFX kit; Life Technologies, USA (1:400 dilution)] for 2 h at room temperature in the dark. After washes in PBS, sections were stained with 4’,6 diamidino-2- phenylindole and mounted. Images were captured on a ZIESS LSM 700 confocal microscope and fluorescence intensity estimated in defined fields using Image J software package [25 ].

N1E-115 cells were grown on glass coverslips coated with laminin in 35-mm tissue culture dishes and treated with the respective drugs diluted in serum-free medium; the cells were then fixed, labeled and visualized by immunofluorescence microscopy. Cells bearing neurite-like structures with a length of at least one cell diameter were identified by immunofluorescence microscopy using an α-tubulin antibody. At least 200 cells were counted for each condition, and the experiments were repeated three times. The images were taken using a Ziess LSM 700 confocal microscope (Oberkochen, Germany).

Colon sections (5μm) were deparaffinized, rehydrated through a series of washes in graded ethanol and water, followed by an antigen retrieval step (by boiling the sections in 10 mM sodium citrate buffer, pH 6.0 for 20 min). Sections were then incubated in blocking solution (5% bovine serum albumin (BSA) + 0.3% Triton X-100 in PBS) for 1 h, followed by incubation overnight at 4°C with primary antibodies [p-ERK1/2, p-Akt (1:50 dilution), p-p38 MAPK and MAS-1R (1:100 dilution); Cell Signaling, USA, and Ang II (1/50 dilution); Novus Biological] diluted in 1% blocking solution. On the following day, sections were washed and incubated with secondary antibody conjugated to Alexa Fluor 555 (Goat anti rabbit SFX kit; Life Technologies,USA, 1:400 dilution) for 2 h at room temperature) in the dark. After washes in PBS sections were stained with 4’,6 diamidino-2- phenylindole and mounted. Images were captured on a ZIESS LSM 700 confocal microscope and fluorescence intensity estimated in defined fields using Image J software package.