Indirect immunofluorescence (IF) was performed as previously described [9 (link)]. The primary and secondary antibodies that were used in these experiments are listed in S1 Table ; a mouse monoclonal anti-α-tubulin served as the positive control. An Olympus BX-51 microscope was used for sample evaluation; micrographs were captured using an Olympus DP72 CCD camera and were analyzed using the Cell^P imaging system (Olympus, Tokyo, Japan).
Cell p imaging system
Manufactured by Olympus
Sourced in Japan
The Cell^P imaging system is a high-performance microscope designed for cell imaging. It provides advanced capabilities for capturing and analyzing images of cells and cellular structures. The system incorporates state-of-the-art optics and imaging technologies to deliver exceptional image quality and resolution. It is a versatile tool suitable for a wide range of cell-based research applications.
Automatically generated - may contain errors
Lab products found in correlation
Dp72 ccd camera, by Olympus (4 mentions)
Bx51 microscope, by Olympus (4 mentions)
Prolong diamond antifade mountant, by Thermo Fisher Scientific (2 mentions)
Digitonin, by Merck Group (1 mentions)
Hoechst 33342, by Thermo Fisher Scientific (1 mentions)
Paraformaldehyde (pfa), by Merck Group (1 mentions)
Triton x 100, by Merck Group (1 mentions)
4 protocols using cell p imaging system
1
Indirect Immunofluorescence for Protein Localization
2
Indirect Immunofluorescence Microscopy of Cell Cultures
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For indirect immunofluorescence (IF), cells were cultivated on coverslips in Petri dishes for 1–2 days (grown to 80–90% confluence), depending on the cell proliferation rate. IF was performed as previously described [29 (link)]. The primary and secondary antibodies used in these experiments are listed in Table 2 . Anti-CP110 antibody was a gift from Dr. Cajanek [30 (link)]. Negative controls were prepared by omitting the primary antibody. After a final wash with PBS, the coverslips were mounted using ProLongTM Diamond Antifade Mountant (Thermo Fisher Scientific, Waltham, MA, USA). An Olympus BX-51 microscope was used for sample evaluation; micrographs were captured using an Olympus DP72 CCD camera and analyzed using the Cell^P imaging system (Olympus, Tokyo, Japan).
3
Indirect Immunofluorescence Microscopy Protocol
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Indirect immunofluorescence (IF) was performed as previously described [3 (link)]. The primary and secondary antibodies that were used in these experiments are listed in Table S3 ; mouse monoclonal anti-α-tubulin served as the positive control. An Olympus BX-51 microscope was used for sample evaluation; images were captured using an Olympus DP72 CCD camera and were analyzed using the Cell^P imaging system (Olympus, Tokyo, Japan). The samples were prepared from at least three independent passages of all examined cell lines, and at least 200 cells were evaluated in each sample. The immunoreactivity and the percentage of cells showing positivity for the examined antigen were determined. Finally, for each cell line, the total immunoscores were calculated for individual antigens by multiplying the percentage of positive cells by the respective immunoreactivity as described previously [69 (link)].
4
Indirect Fluorescence Analysis of Cellular Localization
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Indirect fluorescence analysis was performed as described previously [59 (link)]. The cells on coverslips were fixed with 3% paraformaldehyde (Sigma-Aldrich) at RT for 20 min. The samples were then permeabilized with 0.2% Triton X-100 (Sigma-Aldrich) in PBS at RT for 1 min. For the DOX and LAMP-2 colocalization analysis, 0.2% Triton was replaced with 100 µM digitonin (Sigma-Aldrich), and the cells were permeabilized at RT for 10 min. The primary and secondary antibodies used for indirect fluorescence staining are listed in Table 6 . Coverslips used as negative controls were prepared by omitting the primary antibody. The cell nuclei were counterstained with 0.05% Hoechst 33342 (Life Technologies, Carlsbad, CA, USA). The coverslips with stained cells were mounted using ProLong Diamond Antifade Mountant (Thermo Fisher Scientific, Waltham, MA, USA). For fluorescence evaluation, an Olympus BX-51 microscope was used; the micrographs were captured using an Olympus DP72 CCD camera and analyzed using a Cell^P imaging system (Olympus, Tokyo, Japan).
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