Anti p62 sqstm1primary antibody

Control (untreated or mCNF1‐treated) and CNF1‐treated fibroblasts, seeded on glass coverslips, were fixed with 3.7% paraformaldehyde (Carlo Erba), permeabilized with Triton 0.5% X‐100 (Sigma‐Aldrich, St. Louis) and then incubated at 37°C for 30 min with Tetramethylrhodamine‐isothiocyanate (TRITC)‐phalloidin (Sigma‐Aldrich, cat. n. P1951), diluted 1:100 in PBS for actin cytoskeleton staining. Nuclei were stained for 5 min at RT with 0.2 μg/ml Hoechst 33258 (Sigma‐Aldrich, cat. n. 94,403). To stain mitochondria, cells were incubated with 1 μM Mitotracker Red CMXRos (Invitrogen, Waltham, cat. n. M7512), 30 min before fixation with paraformaldehyde. To stain p62, cells were incubated with Anti‐p62/SQSTM1primary antibody (Sigma‐Aldrich, cat. n. P0067) for 30 min and, following extensive washing, incubated with the appropriate secondary antibody (Alexa Fluor Thermofisher Scientific Invitrogen, cat n. A‐11034). Finally, glass slides were observed with an Olympus BX51/BX52 fluorescence optical microscope (Olympus Corporation of the Americas, Center Valley) equipped with a charge‐coupled device camera (Carl Zeiss). Images were acquired using the IAS 2000 (Delta Systems Inc., Streetsboro) programme.

Using published protocols for immunofluorescence of melanoma transplants [38 (link)], 6 μm cryosections were treated with citrate buffer for antigen retrieval. Tissue auto-fluorescence was quenched with 0.1 M glycine-PBS for 10 min. Sections were then incubated with blocking solution containing 10% goat serum and 1% BSA (30 min) before 1 h staining with the rabbit polyclonal anti-p62/SQSTM1 primary antibody (Sigma-Aldrich, Saint Louis, MO, USA) in blocking solution supplemented with 0.1% saponin. For fluorescent detection Alexa Fluor 546 dye-conjugated anti-rabbit IgG (Life Technologies, Monza, Italy) was used. Tissue sections were rinsed and coverslips added in the mounting medium containing DAPI. At least 5 tissue sections were obtained from each tumour. Images were acquired with a DMI4000 B automated inverted microscope as specified above. Immuno-reactivity was quantified with ImageJ software (http://rsbweb.nih.gov/ij/). In particular, the extent of staining of each section was calculated as integrated optical density, which is equal to the area × average density of image occupied by immune-reactivity and represented in graph.