NCSC-like cells and differentiated cells were stained as described elsewhere (Li et al., 2010 (link)). Cells were fixed with either 4% paraformaldehyde in phosphate buffer solution (PBS) for 20 min at room temperature or in acetone-methanol at −20°C, then permeabilized with 0.5% Triton X-100 for 5 min. After blocking with 5% bovine serum albumin, cells were stained with the primary antibodies listed in Supplementary Table 1 or with isotype-matched IgG at 4°C. Cells were washed 3 times with PBS, and fluorochrome-conjugated secondary antibodies were added and incubated for 1 h at room temperature. Microscopy and photo capture was performed at room temperature on a Nikon E600 upright fluorescence microscope using 20, 40, or 60x objective lenses with a Spot RT Slider digital camera and ImagePro Plus software. Paraffin-embedded skin reconstructs were sectioned and deparaffinized, followed by antigen retrieval, and staining as described above.
E600 upright fluorescence microscope
Manufactured by Nikon
Sourced in Japan
The E600 upright fluorescence microscope is a laboratory equipment designed for imaging and analysis tasks. It features a stable upright design and supports a range of fluorescence imaging techniques. The core function of the E600is to provide a platform for high-quality fluorescence microscopy.
Automatically generated - may contain errors
4 protocols using e600 upright fluorescence microscope
1
Immunofluorescence Staining of NCSC-like and Differentiated Cells
2
Immunofluorescence Staining of NCSC-like and Differentiated Cells
3
Biotin-PEG Nanoparticle Binding Assay
4
Collagen IV Functionalization of PLA Films
Check if the same lab product or an alternative is used in the 5 most similar protocols
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The physico-chemical characterization of the PLA films during the different steps of the functionalization process has already been reported elsewhere [39] (link). Contact angle (CA) measurements were carried out with an optical contact angle device (OCA15, Dataphysics, Germany) before and after col IV functionalization of the PLA films.
Immunofluorescence was used to determine the presence of col IV on the surface of the PLA films. In brief, the samples were blocked for 20 min at room temperature with PBS-Glycine-bovine serum albumin.
After washing with PBS-Glycine (2 × 5 min), the samples were incubated with the primary antibody (Table 2) for 45 min at 37 °C and washed with PBS-Glycine (2 × 5 min). The incubation of the secondary antibody (Table 2) was performed in the dark over 45 min at 37 °C. The samples were visualized with a Nikon E600 upright fluorescence microscope (Tokyo, Japan). Different controls were performed by incubation of either only primary or secondary antibodies.
For col IV quantification, a Micro BCA™ Protein Assay Kit was used, following the manufacturer's protocol. The experiment was conducted in triplicate with six replicas per experiment.
Immunofluorescence was used to determine the presence of col IV on the surface of the PLA films. In brief, the samples were blocked for 20 min at room temperature with PBS-Glycine-bovine serum albumin.
After washing with PBS-Glycine (2 × 5 min), the samples were incubated with the primary antibody (Table 2) for 45 min at 37 °C and washed with PBS-Glycine (2 × 5 min). The incubation of the secondary antibody (Table 2) was performed in the dark over 45 min at 37 °C. The samples were visualized with a Nikon E600 upright fluorescence microscope (Tokyo, Japan). Different controls were performed by incubation of either only primary or secondary antibodies.
For col IV quantification, a Micro BCA™ Protein Assay Kit was used, following the manufacturer's protocol. The experiment was conducted in triplicate with six replicas per experiment.
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