Abc avidin biotin dab detection kit

Teratomas were isolated and fixed at 4 °C overnight with fresh 4% paraformaldehyde in phosphate-buffered saline (PBS) and then embedded in paraffin. Five-micrometer paraffin sections were prepared using a microtome and stained with hematoxylin and eosin. For immunohistochemical staining, the sections were deparaffinized and rehydrated using the standard protocol. Antigen retrieval was performed in a solution (10 mM trisodium citrate, pH 6.0/0.05% Tween-20) by boiling for 10 min in a microwave oven. The tissue sections were incubated with blocking solution (10% goat serum, 1% bovine serum albumin/Tris-buffered saline (BSA/TBS) for 1 h at room temperature and reacted with anti-Taz antibody (Sigma-Aldrich) at 4 °C overnight and corresponding biotinylated secondary antibody diluted 1:500 in 1% BSA/TBS at room temperature for 1 h. The slides were incubated in 0.3% hydrogen peroxide in TBS for 15 min to block endogenous peroxidase. An ABC avidin-biotin-DAB detection kit (Vector Labs, Burlingame, CA, USA) was then used for the detection and visualization of staining according to the supplied protocol. Finally, slides were counterstained with hematoxylin and dehydrated for coverslip mounting. Images were obtained using Observer.Z1 or Imager.M1 (Zeiss).

Tissues derived from mouse studies were fixed in formalin overnight at 4°C and mounted in paraffin using the Tissue‐Tek VIP 6 Vacuum Infiltration Processor (Sakura). Sections (5–8 μm) were cut using a Microm HS355S microtome (Thermo Fisher Scientific) and incubated at 56°C for 1 h. For fixed‐frozen sections, formalin‐fixed tissues were embedded in OCT compound (Sakura) and frozen at −80°C. Sections (8–10 μm) were cut using a Microm HM 525 cryotome (Thermo Fisher Scientific). For vimentin staining of mouse lung tissues or phosphorylated c‐Jun (Ser73) staining of patient‐matched primary tumor and lung metastasis samples, paraffin‐ or OCT‐embedded sections were rehydrated, treated with 3% hydrogen peroxide for 10 min and antigen retrieval was performed using citrate buffer (pH 6.0, Vector Laboratories) at 100°C for 20 min. Then, sections were incubated with blocking buffer, followed by incubation with respective primary antibodies (human vimentin: Leica, clone SRL33, 1:400; p‐c‐Jun (Ser73): Cell Signaling, clone D47G9, 1:50). Corresponding anti‐mouse or anti‐rabbit IgG biotinylated secondary antibodies and an ABC avidin–biotin–DAB detection kit (all from Vector Laboratories) were used for detection and visualization of antigens according to manufacturer's instructions. Sections were analyzed using a Zeiss AxioPlan microscope.

Immunohistochemistry (IHC) was performed on 5 µm sections. Antigen retrieval was carried out with boiling target retrieval solution (Dako, S169984) for 30 min, and samples were blocked in TNB buffer (0.1 M Tris-HCL, pH 7.5, and 0.15 M NaCl with 0.5% w/v blocking reagent (Perkin Elmer, FP1020)) for 1 h at room temperature. Samples were incubated with primary antibodies against Ki67 (Sigma, 275R-18) for 1 h at room temperature. Corresponding anti-rabbit biotinylated secondary antibodies and the ABC avidin–biotin–DAB detection kit (Vector laboratories, PK-6100) were used according to manufacturer’s instructions. Sections were counterstained with Mayer’s hematoxylin solution (Sigma-Aldrich, 51725) for 1 min, dehydrated using increasing concentrations of ethanol, and mounted with Cytoseal XYL (ThermoFisher Scientific, 12502736). Images were acquired using a 20× magnification on TissueFAX system (Tissuegnostic) and processed with FIJI (ImageJ) or ZEN software.