Vector novared substrate kit

De-identified human liver specimens from liver explants were obtained from the Liver Center Tissue Bank at the University of Kansas Medical Center. All studies using human tissue samples were approved by the Human Subjects Committee of the University of Kansas Medical Center.
Immunohistochemistry were performed as previously described [18 (link)]. After deparaffinization and rehydration, antigen retrieval was achieved by heating in a pressure cooker for 5 min in 10 mM of sodium citrate (pH 6). Peroxidase activity was blocked by incubation in 3% H₂O₂ for 10 min. Sections were rinsed three time in PBS/PBS-T (0.1% Tween-20) and incubated in Dako Protein Block (Dako, Agilent Technologies, Santa Clara, CA) for 10 min. After removal of blocking solution, slides were placed into a humidified chamber and incubated overnight with primary antibodies in blocking buffer (3% normal goat serum in PBS) and incubated over night at 4 °C. After washing, slides were covered with SignalStain Boost IHC Detection Reagent (Cell Signaling Technologies, Boston, MA) for 30 min at room temperature. After washing two times with PBS-T, the Substrate-Chromgen Solution (VECTOR NovaRED, Substrate Kit, Vector Laboratories, Burlingame, CA) was applied, slides were incubated 5–10 min and counterstained with Hemtoxylin. Images were acquired using a Nikon Eclipse 80i microscope (Nikon Americas Inc., Melville, NY).

Immunohistochemistry was performed as previously described (Li et al., 2018 (link); Zhao et al., 2019 (link)). After deparaffinization and rehydration, antigen retrieval was achieved by heating in a pressure cooker for 5 min in 10 mM of sodium citrate (pH6). Peroxidase activity was blocked by incubation in 3% H₂O₂ for 10 min. Sections were rinsed three time in PBS/PBS-T (0.1% Tween-20) and incubated in Dako Protein Block (Dako, Agilent Technologies, Santa Clara, CA) for 10 min. After removal of blocking solution, slides were placed into a humidified chamber and incubated overnight at 4°C with primary antibodies in blocking buffer (4% normal goat serum in PBS). After washing, slides were covered with SignalStain Boost IHC Detection Reagent (Cell Signaling Technologies, Boston, MA) for 30 min at room temperature. After washing two times with PBS-T, the substrate-chromgen solution (VECTOR NovaRED, Substrate Kit, Vector Laboratories, Burlingame, CA) was applied, and the slides were incubated for 5–10 min and counterstained with hemtoxylin. Images were acquired using a Zeiss Axiolab 5 Digital Lab Microscope (Carl Zeiss AG, Jena, Germany).

Dissected tumor tissues and metastatic lymph nodes from tumor-bearing nude mice were fixed with 4% PFA and embedded in paraffin. 5-μm thick sections were deparaffinized with xylene and rehydrated in graded ethanol. For the purpose of antigen retrieval, the sections were incubated in sodium citrate buffer (10mM, pH 6.0) at 95°C for 20 min. After blocking with 2.5% goat serum in PBS, the sections were incubated overnight at 4°C with primary antibodies (1:200) for vimentin (rabbit IgG; Cell Signaling Technology, Danvers, MA, USA), ALDH1 (rabbit IgG; Cell Signaling Technology), or PDK-1(rabbit IgG; Enzo Life Sciences, Inc., Farmingdale, NY). For the subsequent steps, sections were stained with VECTASTAIN Universal ABC Elite Kit (PK-7200; Vector Labs, Burlingame, CA) and colors were developed using VECTOR NovaRED Substrate Kit (SK-4000; Vector Labs) according to the manufacture's procedures and briefly counterstained with hematoxylin and observed under a light microscope with a digital camera.

Human Malignant melanoma, metastatic malignant melanoma and nevus tissue array (ME1004a and ME1004b, US Biomax, Rockville, MD, USA) were used for immunohistochemistry staining. Antigen retrieval was achieved by microwaving arrays immersed in 1 liter Tris-EDTA buffer (10 mM Tris base, 1 mM EDTA solution, 0.05% Tween 20, pH 9.0) for 30 min. Tissue arrays were treated with 1% H₂O₂ in water for 15 min and blocked in 10% BSA in Tris-buffered saline for 2 h at room temperature. Anti-Mov10 (1:200) in 10% BSA/Tris-buffered saline or anti-FASN (1:500) in 1% BSA/Tris-buffered saline were applied and incubated at 4 °C overnight. Then, arrays were incubated with biotinylated anti-rabbit (1:500, Vector Lab.) for Mov10 or rabbit anti-mouse (1:200, Vector Lab., Burlingame, CA, USA) for FASN for 1 h at room temperature followed by being incubated with VECTASTAIN ABC kit (PK-4000, Vector Lab.) for 30 min. Mov10 and FANS were visualized by Vector Novared substrate kit (SK-4800, Vector Lab.). The arrays were counterstained with hematoxylin for 1.5 min.