Three-dimensional localization of p62 and ubiquitin was analyzed by double immunofluorescence staining and followed by reconstruction as previously described with minor modifications20 (link). One-μm FFPE tissue sections were cut, dewaxed, rehydrated and pretreated with Target Retrieval Solution pH9 (#S2367, Dako) for 20 min at 97 °C.
For double labelling immunofluorescence the primary antibodies anti-p62 (#BML PW9860, Enzo, Life Sciences, NY, USA, diluted 1:250 for 60 min at RT) and anti-ubiquitin (#NB300-130, Novus, Littleton, CO, diluted 1:100 for 30 min at RT) were used. Secondary antibodies used were Alexa Fluor 488-conjugated donkey anti-rabbit IgG (#A21206, Thermo Fisher Scientific) and Alexa Fluor 594-conjugated goat anti-mouse IgG1 (#A21125, Thermo Fisher Scientific). Anti-p62 antibody was labelled with Alexa Fluor 488 and anti-ubiquitin antibody with Alexa Fluor 594. After 1:100 dilution they were incubated for 60 min at RT. DNA was stained with DAPI and image analysis was performed as described above.
For double labelling immunofluorescence the primary antibodies anti-p62 (#BML PW9860, Enzo, Life Sciences, NY, USA, diluted 1:250 for 60 min at RT) and anti-ubiquitin (#NB300-130, Novus, Littleton, CO, diluted 1:100 for 30 min at RT) were used. Secondary antibodies used were Alexa Fluor 488-conjugated donkey anti-rabbit IgG (#A21206, Thermo Fisher Scientific) and Alexa Fluor 594-conjugated goat anti-mouse IgG1 (#A21125, Thermo Fisher Scientific). Anti-p62 antibody was labelled with Alexa Fluor 488 and anti-ubiquitin antibody with Alexa Fluor 594. After 1:100 dilution they were incubated for 60 min at RT. DNA was stained with DAPI and image analysis was performed as described above.
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