Immunofluorescence Analysis of Lysosomal and Autophagic Markers in hDF Cells
Corresponding Organization : National Research Council
Other organizations : University of Campania "Luigi Vanvitelli", UK Dementia Research Institute, Cardiff University, Sapienza University of Rome, University of Oxford
Variable analysis
- Starvation treatment (1 h in HBSS)
- Localization and expression of LAMP1, LAMP2, p62, LC3B, TMEM175, GlcCer, GM1
- HDF cells (20,000 cells in 24-well plates on coverslip)
- Complete medium for 24 h
- Fixation with paraformaldehyde (PFA) 4%
- Blocking buffer (1 × PBS/0.1% triton/5% serum) for 15 min
- Primary antibody incubations at 4 °C overnight
- Secondary antibody incubations at RT for 1 h
- Hoechst H3570 (1:1000) for nuclei visualization
- Not explicitly mentioned
- Not explicitly mentioned
Annotations
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