Ki67 sp6

Antibodies for immunostaining and western blotting were used as per the manufacturer’s recommendations. The following antibodies were used within this study: β-tubulin (Cell Signalling Technology, Cat # 2146 (1:1000)), Cleaved Caspase-3 [Asp175] (Abcam, Cat # ab49899 (WB 1:500, IHC 1:1500)), CDKN2A/p19ARF (Abcam, Cat # ab80 (1:1000)), cMYC [Y69] (Abcam, Cat # ab32072 (WB 1:1000, IHC 1:500)), Cyclophilin B (Cell Signalling Technology, Cat # 43603 (1:1000)), GFAP [GA5] (Millipore, Cat # MAB3402 (1:1000)), GFP (Abcam, Cat # ab13970 (1:1000)), HSF1 (Abcam, Cat # ab61382 (1:1000)), HSP70 (Abcam, Cat # ab2787 (1:1000)), HSP90 [EPR16621] (Abcam, Cat # ab203085 (1:1000)), Ki67 [SP6] (Abcam, Cat # ab16667 (1:2000)), n-MYC [NCM II 100] (Abcam, Cat # ab16898 (1:250)), NPR3 (Abcam, Cat # ab97389 (1:250)), Olig-2 (Millipore, Cat # AB9610 (1:1000)), OTX2 (R&D Systems, Cat # AF1979 (1:500)), p21 (Abcam, Cat # ab109199 (1:1000)), Synaptophysin [SY38] (Millipore, Cat # MAB5258 (1:500)), TUJ1 (Covance, Cat # MMS-435P (1:500)), β-actin (Santa Cruz Biotechnology, Cat # sc-47778 (1:1000)), CDKN2A/p16INK4A (Abcam, Cat # ab211542 (1:1000)), anti-mouse IgG secondary HRP (VWR, Cat # NXA931 (1:1000)), anti-rabbit IgG secondary HRP (GE Healthcare, Cat # NA934 (1:1000)), and Lamin B1 (Abcam, Cat # ab16048 (1:1000)).

Immunohistochemical (IHC) staining was performed after chemical dewaxing of two 3 μm thick sections using antibodies against Ki-67 (SP6) (Abcam, Cambridge, UK) and cleaved caspase-3 (Cell Signaling, Danvers, MA, USA). Ki-67 and cleaved caspase-3 stainings are indicators for proliferation and apoptosis indexes, respectively. IHC staining was carried out by adding 100 μL of DAB+chromogen diluted at 1:50 in substrate buffer [EnVision+ Dual Link System-HRP (DAB+)] for 10 min. Finally, tissue specimens were washed in phosphate buffer saline (PBS), the nuclei counterstained with hematoxylin and mounted using Eukitt® for microscopic examination. Positively stained melanoma cells, in which Ki-67 and cleaved caspase-3 highlight in brown the nucleus and the cytoplasm respectively, were quantified using ImageJ software (National Institutes of Health, Bethesda, MD, USA).

Immunohistochemistry was performed as previously described [28] (link). Briefly, specimens were rehydrated, endogenous peroxidase activity blocked, and incubated either with anti-F4/80 (BM8) for 45 min or anti-Gr-1 (RB-8C5), anti-Ly6C (HK1.4), anti-Ly6G (1A8, BioLegend, CA, USA) and anti-IL-17a (Abcam, England) overnight at 4 C, or Ki67 (SP6, Abcam) for 2 h. Labeling was visualized via HRP-conjugated secondary antibody with DAB precipitate (Abcam). Positive labeling was imaged and quantified as previously described [28] (link).