TMM gels with PC12 cells were incubated in Oregon Green Phalloidin to label cytoskeletal elements. For DRG axonal growth, the tissue was incubated in 4% Donkey serum for 1 h, followed by incubation with a mouse anti-β tubulin III antibody (1:400; Sigma Aldrich) overnight at 4 °C. The gels were then incubated with Cy2-conjugated donkey anti-mouse IgG (1:400; Sigma Aldrich) and rinsed. Long-distance water immersion objectives on a Zeiss confocal microscope (Zeiss Axioplan 2 LSM 510 META) were used to evaluate the cellular staining and axonal growth directly within the hydrogel microchannels.
Axioplan 2 lsm 510 meta
Manufactured by Zeiss
The Zeiss Axioplan 2 LSM 510 META is a laser scanning microscope system. It is designed for high-resolution imaging and analysis of biological samples. The system features a confocal optical design and a range of laser excitation wavelengths to enable advanced fluorescence imaging applications.
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Lab products found in correlation
2 protocols using axioplan 2 lsm 510 meta
1
Assessing Cytoskeletal and Axonal Growth
2
Immunofluorescence Assay for Axonal Growth
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Fixed DRGs were permeabilized in 0.5% PBS-Triton ×100 for 5 minutes. Non-specific staining was blocked with 4% normal donkey serum for 1 hour, followed by incubation with a mouse anti-β tubulin III antibody (1:400; Sigma-Aldrich, St. Louis, MO) overnight at 4 °C. After rinsing, the tissue was incubated for 1 hour with a Cy2-conjugated donkey anti mouse antibody (1:400; Sigma-Aldrich, St. Louis, MO). The stained tissue was imaged on a Zeiss confocal microscope (Zeiss Axioplan 2 LSM 510 META). Axonal growth was estimated from 3 DRG samples per treatment. Z-stacks were imaged at 20X magnification (20 images each at 15.4 μm slice thickness) and individual axons were traced using ImageJ software measuring from the edge of the DRGs to the axon terminals (Fig. 1D–F ).
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