Protein block serum free

Skin sections obtained from the nose of a Caucasian female were fixed in acetone at −20 °C for 10 min and treated with 0.1% Triton X-100/PBS for 5 min. The skin sections were incubated with Protein Block serum-free (Agilent Technologies) for 30 min, then with the primary antibodies against keratin/cytokeratin (mouse monoclonal (AE-1/AE-3), original solution, Nichirei, Tokyo, Japan) or RNase 7 (rabbit polyclonal, 1:50, Cloud‐Clone Corp., Houston, TX, USA) for 1 h at 20–25 °C, and finally with anti-rabbit IgG (donkey polyclonal, Alexa Fluor 555, 1:1000, Thermo Fisher Scientific) or anti-mouse IgG (goat polyclonal, Alexa Fluor 647, 1:1000, Thermo Fisher Scientific) for 30 min. Samples were mounted on a glass slide and imaged using fluorescence microscopy (BZ-X710, Keyence, Osaka, Japan).

Four-micrometer-thick sections from paraffin-embedded block samples obtained from primary lesions before starting primary treatment were deparaffinized in xylene and rehydrated in a graded series of alcohol. Epitope retrieval was achieved by heating to 100 °C for 10 min in 1 mM EDTA buffer (pH 8.0). Endogenous peroxidase activity was quenched by incubating the sections in 0.3% H₂O₂ in methanol for 20 min at room temperature. A SAB-PO Kit (Nichirei Biosciences Inc., Tokyo, Japan) was used to detect immunoreactivity to PD-L1 according to the manufacturer’s protocol. After blocking non-specific reactions with 10% goat serum, the sections were incubated with primary antibodies for 1 h at room temperature. A rabbit monoclonal anti-PD-L1 antibody (E1L3N^®, Cell Signaling Technology, Danvers, MA, USA) was used at a 1:200 dilution with Protein Block Serum-Free (Dako; Agilent Technologies, Inc., Santa Clara, CA, USA). Positive PD-L1 expression was defined as a stained cell membrane by immunohistochemistry (Figure 1).

IHC evaluation of fibulin-3 levels was performed on tumor samples collected from the in vivo experiment illustrated in the work by Baroni et al., 2016 (11) (6 samples per experimental condition). Tissue sections were deparaffinised, rehydrated and heated for 5 min at 95 °C in citrate buffer (4:1 sodium citrate (10 mM, pH 8) and citric acid (5 mM); final pH 6). Peroxidase blocking was achieved with 15 min incubation in 80% methanol and 3% hydrogen peroxide. Sections were then incubated with Protein Block Serum-Free (Dako products, Agilent Technologies, Santa Clara, CA, USA) in BSA 1%. Slides were then incubated at room temperature for 1h with a mouse monoclonal anti-fibulin-3 antibody (1:100, clone: C-3, catalog number: sc-365224, Santa Cruz Biotechnology, Dallas, TX, USA) and then with Biotinylated anti-mouse secondary antibody (1:100, Dako) for 45 min. Antibodies were diluted in “Dako real antibody diluition” (Dako products, Agilent Technologies, Santa Clara, CA, USA). Follows HRP-conjugated streptavidin (1:300) for 30 min, DAB (1:50 in HRP substrate buffer) staining for 5 min and mayer’s hematoxylin counterstaining for 10 s. Sections were finally dehydrated and mounted. A positivity score ranging from 0 to 2 was assigned to each tumor, having 0 for no signal, 1 for intermediate positivity and 2 for high positivity.