Dab 3 3 diaminobenzidine

Paraffin-embedded EOC tumors were analyzed with anti-Axl and anti-p130Cas Abs. IHC was performed after deparaffinization, as described [38 (link)]. The primary goat anti-human Axl Ab was diluted 1:25 and the primary mouse anti-human p130Cas Ab was diluted 1:400. Incubation with each of the primary antibodies was performed overnight at 4°C, then the slides were incubated for 30 min at room temperature with the secondary biotinylated antibodies diluted 1:200. The slides were washed with PBS, and the peroxidase activity was revealed by incubating sections in DAB (3–3′diaminobenzidine) (DAKO, Denmark) for 5 min. After washing with water, sections were counterstained with Gill's hematoxylin solution for 5 sec.

TMA, which was constructed in previous studies, was used in this study.²⁷, ²⁸ The TMA blocks were cut to 5‐μm thickness with a rotary microtome. After sectioning, the TMA sections were deparaffinized with xylene and dehydrated in serially graded ethanol to distilled water. Then, antigen retrieval was performed by incubating TMA sections using a steam pressure cooker (Pascal; Dako, Carpinteria, CA) in heat‐activated antigen retrieval buffer at pH 6.0 (Dako) for anti‐NANOG and at pH 7.8 for anti‐phospho AMPKα (Thr172). The sections were treated with 3% H₂O₂ solution in methanol for 10 min to block the endogenous peroxidase activity. After rinsing the slides, they were stained with an anti‐NANOG antibody (rabbit antibody, clone#4903S, 1:200; Cell signalling Technology, Inc., Danvers, MA) for 1 hour and anti‐pAMPKα antibody (rabbit antibody, clone#2535, 1:52; Cell signalling Technology, Inc.) for 32 min at room temperature. Subsequently, the antigen–antibody reactions were visualized by using Envision⁺ Dual Link System‐HRP (Dako) and DAB⁺ (3, 3′‐diaminobenzidine; Dako) for 10 min. The stained sections were dehydrated and counterstained with haematoxylin and mounted in Faramount aqueous mounting medium (Dako). Appropriate negative and positive controls were included.

Immunohistochemistry was performed on cells which were: (i) previously removed from culture flasks or from 3D membrane using the Trypsin 0.25% and fixed on slides; (ii) fixed on the membrane fibers in formaldehyde or ice cold acetone. Next, the Universal Dako REAL EnVision detection system, peroxidase/DAB+, rabbit/mouse (Dako, Copenhagen, Denmark) were used. Then, cell samples were incubated with primary antibodies (CD44, CD90, CD105, type I, II and X collagen, vimentin) for 1.5 h at room temperature and aggrecan, for 24 h at 4°C. The antigen–antibody reaction was visualized by DAB (3,3 diaminobenzidine) (Dako, Copenhagen, Denmark) as a chromogen for 4 min at room temperature and counterstained with hematoxylin and mounted. The samples incubated without primary antibodies were used as negative controls.

The collected specimens of the mandible (1.0 cm long, 1.0 cm wide and 1.0 cm thick bone blocks) were fixed in neutral formalin for one week, decalcified in chelaton for one month, dehydrated through a series of 70–100% ethanol solutions and embedded in paraffin. The specimens were then serially sectioned at 7 μm thickness in the sagittal plane, mounted on slides and prepared for histological (haematoxylin and eosin staining) and immunohistochemical analysis. The physiological bone sections from the left side of the mandible were used as reference control samples.
An immunohistochemical reaction was performed to demonstrate the presence of collagen I using a primary rabbit polyclonal anti-collagen I antibody (Abcam, ab233080) and a secondary DB DET SYS kit, the DB detection kit—rabbit/mouse dual system (Biotech). DAB (3,3′-diaminobenzidine) (DAKO) was used to visualize the reaction. Finally, the cell nuclei were stained with acidic Mayer’s haematoxylin.