Envision flex epitope unmasking solution

Immunohistochemical analysis of p62, LC3, and Beclin1 markers was performed on TMA sections with Autostainer Link 48 (Agilent, Glostrup, Denmark). EnVision FLEX epitope unmasking solution, pH 6.1 (K8005, Agilent), was used for LC3B antibody epitope unmasking. EnVision FLEX epitope unmasking solution, pH 9.0 (K8004, Agilent), was used for p62 and Beclin1 antibody epitope unmasking. Immunohistochemical analysis was performed with the visualization system EnVision FLEX (Agilent). The following primary antibodies were used: polyclonal rabbit anti-human p62 antibody (ab155686, Abcam, Cambridge, UK) at 1:500 dilution; monoclonal recombinant rabbit anti-human Beclin-1 antibody (EPR20473, Abcam) at 1:50 dilution; and polyclonal rabbit anti-human LC3B antibody (ab4839zhen, Abcam) at 1:200 dilution. The percentage of positive cells was recorded. The microscope magnification was x400. For immunohistochemical interpretation, we used the following score: negative (0) expression – without positive cells or with a single positive cell (<1%); low (1+) expression – less than 10% positive cells; moderate (2+) expression – 10%-50% positive cells; and strong (3+) expression – more than 50% positive cells (18 (link)).