Lsab hrp kit

Sections (4 μm) mounted on poly-L-lysine-coated slides were incubated for 30 min at 60 °C, deparaffinised by standard methods, and placed in 0.05 M Tris-HCI buffer, pH 7.2. Antigen retrieval was performed for 20 min in 10 mm sodium citrate buffer (pH 6) heated at 95 °C in a steamer, followed by cooling for 20 min. After blocking endogenous peroxidase activity with 0.3 % aqueous hydrogen peroxide for 5 min, the primary polyclonal rabbit anti-TGF-β1 antibody (DAKO, Carpinteria, CA, USA) was incubated with the sections at a final dilution of 2 mg/mL for 30 min. A control slide was incubated with Tris-HCI buffer substituted for the primary antibody. The DAKO LSAB™ kit/HRP was used for the detection of immunostaining. The sections were counterstained with Mayer’s haematoxylin. The staining intensity of TGF-β1 was semiquantitatively scored as negative, weak or moderate/strongly positive. A complete negative staining was scored as negative. A weak staining was defined as a minimal but unequivocal staining in less than 10 % of tumour cells. Stronger or more extensive staining was scored as moderate (10–50 %) or strong (≥50 %) positive expression. For statistical analysis, patients with weak and moderate/strong staining were lumped together as the TGF-β1-positive group in comparison with those with TGF-β1-negative tumours.

For histologic analysis, paraffin-embedded pancreatic sections were stained with hematoxylin and eosin. Immunohistochemistry reactions were performed on paraffin-embedded sections. First, the sections were incubated with Peroxidase-Blocking Reagent (DAKO Cytomation, Fort Collins, CO, USA) followed by incubation with PBS + 1% bovine serum albumin (Sigma-Aldrich) and Triton X-100 (Sigma-Aldrich) to prevent unspecific staining. Next, rabbit monoclonal anti-mouse insulin antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA) was applied to the sections, followed by incubation with the LSAB™ + Kit/HRP (DAKO Cytomation). The slides were stained with diaminobenzidine according to the manufacturer’s instructions (DAKO Cytomation). Finally, the sections were counterstained with Harris hematoxylin and analyzed by light microscopy.

For histologic analysis, pancreata were removed, fixated in 10 % neutral buffered formalin, and embedded in paraffin and the sections (5 μm) were stained with hematoxylin and eosin (H & E). Immunohistochemistry reactions were performed on formalin-fixed or frozen Tissue-Tek O.C.T (Sakura Finetek, Zoeterwoude, the Netherlands) tissue sections. First, sections were incubated with Peroxidase-Blocking Reagent (DAKO Cytomation, Fort Collins, CO, USA) to block endogenous peroxidase. The slides were then incubated with a blocking solution containing PBS/bovine albumin serum 1 % (Sigma)/Triton X-100 (BioRad, Richmond, CA, USA) to prevent unspecific staining. Next, rabbit monoclonal anti-mouse insulin antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA) or rabbit anti-mouse Ki-67 antibody (Abcam, Cambridge, UK) were applied to the sections, followed by incubation with LSAB™ + Kit/HRP (DAKO Cytomation). The slides were stained with diaminobenzidine according to the manufacturer’s instructions (DAKO Cytomation). Finally, the sections were counterstained with Harris hematoxylin and analyzed under light microscopy.

For immunohistochemical studies with a DAKO LSAB kit (DAKO A/S, Glostrup, Denmark), 4 μm-thick tissue sections were deparaffinized, rehydrated and incubated with 3% H₂O₂ in methanol for 15 minutes at room temperature to eliminate endogenous peroxidase activity. Antigen retrieval was performed by placing the slides in 0.01 M sodium citrate buffer (pH 6.0) at 95°C for 20 minutes. After overnight incubation at 4°C with primary antibody against Ezrin (Cell Signaling, USA), sections were treated according to standard immunoperoxidase methods using a streptavidin-biotin peroxidase complex kit (LSAB+Kit/HRP, DAKO). The peroxidase reaction was developed with 3, 3′-diaminobenzidine (DAB), and then counterstained with Mayer's hematoxylin. Rabbit IgG isotope used as a negative control and positive tissue sections were processed omitting the primary antibody as a further negative control. All slides were evaluated independently by two pathologists without prior knowledge of clinical outcome.

p21, p53, and c-myc were detected by immunohistochemical method. All primary antibodies and mouse monoclonal antibodies were purchased from Dako (Hamburg. Germany). Immunohistochemical staining was performed by the enhance labeled polymer system (ELPS) method. After overnight incubation at 4°C with anti-p21, p53, and c-myc antibody, sections were treated according to standard immunoperoxidase methods using a streptavidin biotin peroxidise complex kit (LSAB + Kit/HRP; Dako). The peroxidise reaction was then developed with diaminobenzidine (Dako). Negative control sections were subjected to the same procedure except that the first antibody was replaced by phosphate buffer saline (PBS) [20 (link)].

Serial cuts were performed on the same tissue samples used for H&E staining. Versican, adiponectin, Adipo R1, CD44, and perilipin expression were studied by means of immunohistochemistry. Briefly, hATT and hATN microtome slides were first deparaffinized, and then a heat-mediated antigen retrieval, endogenous peroxidase blocking and nonspecific tissue blocking were performed. Slides were then incubated with the different primary antibodies at 4 °C, and after that with an anti-rabbit biotinylated secondary IgG antibody. Finally, slides were incubated with peroxidase-conjugated streptavidin. Peroxidase reaction was performed with chromogen 3,3′-diaminobenzidine (DAB) (DAKO LSAB + Kit, HRP). Hematoxylin counter stain was performed. Serial cuts incubated in the absence of primary antibody were used as negative controls. Images were taken with a Nikon Eclipse E200 Microscope fitted with a Micrometric SE Premium (Nikon Corp., Japan) digital still camera at 100x and 400x magnifications. DAB staining quantification in the three tissue types was performed in 8–10 fields of each preparation as mentioned above.