For analysis of neural differentiation of ADSC, differentiated cells were fixed with 4% paraformaldehyde, and incubated with 10% goat serum to prevent nonspecific antibody binding. The cells were incubated overnight at 4°C with several rabbit polyclonal anti-human Nestin, MAP2, RIP, and GFAP antibodies (Sigma, St. Louis, MO, USA, final dilution 1∶500). After extensively washing in PBS, the cells were then incubated for 30 min with Alexa fluor 488 conjugated secondary antibodies (1∶400–500; Invitrogen, Carlsbad, CA, USA). Controls in which primary antibodies were omitted or replaced with irrelevant IgG resulted in no detectable staining. Specimens were examined using a Leica TCS SP2 laser scanning microscope equipped with three lasers (Leica Microsystems, USA). Immunocytochemical studies were repeated at least three times.
Tcs sp2 laser scanning microscope
Manufactured by Leica
Sourced in United States
The TCS SP2 is a laser scanning microscope manufactured by Leica. It is designed to capture high-resolution images of samples by using a focused laser beam to scan the specimen and detect the emitted fluorescence or reflectance. The instrument provides the core functionality of laser scanning microscopy without extrapolation on its intended use.
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Lab products found in correlation
Gfap antibody, by Merck Group (1 mentions)
Three lasers, by Leica (1 mentions)
Alexa fluor 488 conjugated secondary antibody, by Thermo Fisher Scientific (1 mentions)
Az100 stereo zoom microscope, by Nikon (1 mentions)
Vimentin antibody, by Merck Group (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (1 mentions)
Tcs sp2 microscope, by Leica (1 mentions)
Alexa fluor 488 conjugated rabbit igg, by Thermo Fisher Scientific (1 mentions)
Mouse anti lamp1 antibody, by Abcam (1 mentions)
Cy3 labeled goat anti rabbit igg secondary antibody, by Wuhan Servicebio Technology (1 mentions)
5 protocols using tcs sp2 laser scanning microscope
1
Immunocytochemistry of Differentiated ADSCs
2
Microscopy Imaging of Neuronal Growth
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Bright field and conventional fluorescent images were acquired with a Nikon AZ100 stereo zoom microscope using 1× and 2× objectives (Nikon, Melville, NY), while confocal images were taken using a Leica TCS SP2 laser scanning microscope and 20× objective (Leica Microsystems, Buffalo Grove, IL). Confocal z-stacks were acquired through the maximum depth of visible neurite growth with thicknesses ranging between 55-65 μm imaged over 20 slices, each 512 × 512. Image processing was performed using ImageJ (National Institutes of Health, Bethesda, MA). For color coding depth in confocal z-stacks, the Z Code Stack function with a Rainbow LUT was applied using the MacBiophotonics Plugin package for ImageJ. Projections of z-stacks were taken as maximum intensity projections. V3D-Viewer software (Janelia Farm Research Campus, Howard Hughes Medical Institute, Ashburn, VA) allowed 3D rendering and visualization of the confocal z-stack images.
3
Confocal Microscopy of Cell Adhesion
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Confocal laser scanning microscopy was performed using a Leica TCS SP2 microscope (Leica Microsystems) as previously reported [31 (link)]. Briefly, cells were grown on 10 mm glass coverslips in 24-well culture plates. After methanol fixation for 10 min, cells were incubated with an E-cadherin antibody (1:100, Invitrigen) or Vimentin antibody (1:100, Sigma), followed by incubation with goat-anti-mouse-Alexa 594 conjugated antibodies (1:500, Invitrogen). Cell nuclei were counterstained with DAPI (1:1000, Invitrogen). Confocal image series were recorded on a LEICA TCS SP2 laser scanning microscope.
4
Immunostaining of Eag1 Channels in Cells
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Cells grown on coverslips were rinsed in ice-cold D-PBS (PBS with 0.9 mM CaCl2, 0.5 mM MgCl2) and fixed for 20 min with 4% paraformaldehyde in PBS at 4 °C. After washing with cold PBS, fixed cells were permeabilized and blocked with a blocking buffer (5% normal goat serum in 20 mM phosphate buffer, pH 7.4, 0.1% (v/v) Triton X-100, and 0.45 M NaCl) for 60 min at 4 °C. Cells were then immunolabeled with the following primary antibodies at 4 °C for 16 hrs: rabbit anti-rEag1 antibody (Alomone; 1:1000), mouse anti-Myc antibody (clone 9E10, 1:100), mouse anti-calnexin antibody (1:200; Cell Signaling), or mouse anti-lamp1 antibody (1:500; Abcam). Alexa Fluor 488-conjugated rabbit IgG and Alexa Fluor 568-conjugated mouse IgG (1:200; Molecular Probes) were used as secondary antibodies. Nuclei were stained with DAPI. After final wash, coverslips were mounted in a mounting medium (4% n-propylgallate, 90% glycerol, 0.1 M carbonate buffer, pH 9.2), and observed using a TCS SP2 laser-scanning microscope (Leica) equipped with a 63x oil immersion objective.
5
Astrocyte Activation and Protein Expression
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The in situ expression of MTDH and the astrocytic marker GFAP was analyzed as described previously [27] . The xed spinal cord was sectioned into 10 mm-thick slices, and probed with rabbit anti-MTDH (1:100, Proteintech), rabbit anti-GFAP (1:800, ServiceBio) and rabbit anti-DAPI (1:800, ServiceBio) primary antibodies, followed by Cy3-labeled goat anti-rabbit IgG secondary antibody (1:500, ServiceBio). The stained sections were observed under the Leica TCS SP2 laser scanning microscope.
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