Dako real envision detection system kit

Cells from each cell line were seeded on coverslips, in 100 mm Petri dishes at 50% confluency, to achieve approximately 70–85% confluency by the end of the 5-day period of PEMF treatment. Cell culture medium was replaced on the third day of treatment. Cells were then fixed using 4% Paraformaldehyde (PFA) for 10 min, at 4 °C, and washed with PBS three times. For the Immunocytochemistry (ICC) process, cell membranes were permeabilized using Triton-X 0.3%/PBS for 15 min at RT, followed by the blocking of non-specific binding sites with goat serum (Abcam ab138478, in 1:40) for 1 h at RT. Cells were then incubated with one of the following primary antibodies for 1 h at RT: anti-biotin (Cat.no: ab201341, dilution 1:300, Hyb-8, Abcam, Cambridge, UK), p21^WAF1/Cip1 (Cat.no: 2947S, dilution 1:400, 12D1, Cell Signaling, Danvers, MA, USA) or Ki67 (Cat.no: ab16667, dilution 1:250, SP-6, Abcam, Cambridge, UK). Signal development was obtained using the Dako REAL EnVision Detection System kit (Cat.no: K5007, Santa Clara, CA, USA) according to the manufacturer’s instructions using 3,3′-Diaminobenzidine (DAB) (brown color). Coverslips were counterstained with hematoxylin and positive cells were counted. Finally, coverslips were sealed and observed under a ZEISS Axiolab5 (Munich, Germany) optical microscope with 20× or 40× objectives [26 (link)].

Immunohistochemical analyses were performed using the Dako REAL™ Envision™ Detection System Kit (DAKO, Glostrup, Denmark, #5007). Tissues were first incubated with primary antibodies (Abs) against IL-1β IL-6, IL-17A, and tumor necrosis factor (TNF)-α (all from Abcam, Cambridge, UK) overnight at 4°C followed by incubation with Dako REAL™ Envision™/HRP for 30 min. The final colored product was developed using a chromogen diaminobenzidine. Three independent, blinded observers assessed all of the histologic scores. Images were taken using a DP71 digital camera (Olympus, Center Valley, PA, USA) attached to a BX41 microscope (Olympus). Positive cells were counted (magnification 400×) with the aid of Adobe Photoshop software and were averaged in three randomly selected fields per tissue section.

Slides were de-waxed in xylene and rehydrated through decreasing ethanol washes, before being incubated in a bleach solution to remove pigment. Antigen-unmasking was performed as previously described (Patton et al., 2005 (link)). with the Dako REAL EnVision Detection System kit (Dako; UK) following manufacturer’s instructions. Slides were incubated overnight at 4°C with the following antibodies: anti-rabbit α-GFP (1:1,500; Cell Signaling Technology) and anti-rabbit α-WT1 (1:25,000; Cambridge Research Biochemicals; UK). An Axioplan II fluorescence microscope (Zeiss; Germany) with a Plan Apochromat objective was used for brightfield imaging of tissue sections. Images were captured using a Qimaging Micropublisher 3.3mp cooled CCD camera and analysed using the IPLab Spectrum software.

Western blotting was performed on the cell‐lines using standard protocols and IHC on TMA using Dako REAL EnVision Detection System kit (K500711; Dako) (Coventry et al., 1995; Hirano, 2012). Antibodies used for IHC are Cdh23 (HPA017232; Sigma) and Ecdh (HPA004812; Sigma). Moreover, Cdh23 N‐terminal (PA5‐43398; Thermo Fisher Scientific, ?Waltham, MA?, USA), β‐catenin (sc‐7963; Santa Cruz, Santa Cruz, CA, USA) and anti‐β‐actin (A1978; Sigma) specific antibodies were used for western blotting. For IF studies, anti‐mouse IGG (H + L), CF594 (SAB4600092; Sigma) and anti‐rabbit IGG (H + L), CF633 (SAB4600141; Sigma) were used as secondary antibodies. Wnt/β‐catenin downstream activated targets were identified using Antibody Sampler Kit #8655 (Cell Signaling Technology, Danvers, MA, USA).