Lsab kit

Immunohistochemistry for myostatin and NF-κB was performed according to a previously described protocol [26 (link)-29 ]. In brief, 4 μm thick sections of paraffin-embedded gastrocnemius skeletal muscle were prepared, deparaffinized, and rehydrated. Next, 3% hydrogen peroxide in methanol was used to block endogenous peroxidase activity. Phosphate-buffered saline (PBS, pH 7.2) was used to wash the sections before and after incubating some of them with myostatin antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA) and incubating others with anti-NF-κB antibody (Santa Cruz Biotechnology), using the dilutions suggested by the manufacturers. The sections were then incubated with a biotinylated secondary antibody (LSAB kit, Dako, Carpinteria, CA, USA) and washed with PBS before being successively incubated with streptavidin horseradish peroxidase (LSAB kit, Dako). They were then rinsed with PBS and treated with 3,3′-diaminobenzidine substrate (Santa Cruz Biotechnology) until the desired color intensity was detected. Finally, the sections were washed with tap water to stop the further reaction. Hematoxylin (Santa Cruz Biotechnology) was used to counterstain the sections. Negative control slides were processed in the absence of a primary antibody. The sections were viewed at 40x under a light microscope (BEL Engineering Company, Italy).

Immunocytochemical staining of cytokeratin pan was performed to prove the epithelial origin of the primary renal tumor cells. Renal tumor cells (5 × 10³ cells/ml) were centrifuged on microscope slides and fixed in 100% ethanol for 10 min. Endogene peroxidase was blocked by a 5 min treatment with peroxidase blocking solution (Dako, Hamburg, Germany). Mouse anti-cytokeratin pan monoclonal antibody (Abcam, Cambridge, UK), diluted 1:200 in antibody-diluent (LSAB + −Kit, Dako, Carpinteria, USA), was incubated for 1 h at room temperature. The secondary biotinylated anti-mouse antibody (Dako) was applied for 30 min at room temperature. After using a horseradish peroxidase-conjugated strepatividin-label (LSAB + −Kit, DAKO) for 30 min, cells were treated with DAB (Diaminobenzidine, Dako) for 10 min and counterstained with Mayer’s Hemalm. For all experiments only cytokeratin positive cells were used.

Immunohistochemistry for β-catenin was performed using the labeled streptavidin-biotin method (LSAB Kit; DAKO, Glostrup, Denmark) to count the number of BCAC [12 (link),23 (link),24 (link)]. The primary antibody for β-catenin (BD Transduction Laboratories, San Jose, CA, USA) was used at a final dilution of 1:1000. Immunohistochemical staining for proliferating cell nuclear antigen (PCNA), which is a G₁-to-S phase marker, and for phospho-nuclear factor-κB (NF-κB) p65 were run on histological sections to estimate cell proliferative activity and NF-κB activity, respectively, in the colonic crypts [11 (link),23 (link)], using the LSAB Kit (DAKO) with primary antibodies, anti-PCNA antibody (a final dilution of 1:100, Santa Cruz Biotechnology, Dallas, TX, USA) and anti-phospho-NF-κB p65 antibody (a final dilution of 1:50, Ser276; Cell Signaling Technology, Danvers, MA, USA). The PCNA-labeling index (%) and positive cell index (%) for phospho-NF-κB p65 were determined based on previous methods [11 (link),23 (link)].

MV inflammatory infiltrates were identified by immunohistochemistry using markers for mononuclear cell subtypes. Cryosections were fixed in −20°C acetone (Fisher Scientific, MA, USA), blocked with 0.3% hydrogen peroxidase (Fisher Scientific, MA, USA) and incubated with Protein Block Serum-Free (DAKO, CA, USA). Primary antibodies anti-CD68 (human, 1:500, DAKO, CA, USA), anti-CD4 (human, 1:30, Fisher Scientific, USA) and anti-CD8 (human, 1:80, Fisher Scientific, USA) were diluted in 5% inactivated normal horse serum (Vector Laboratories, CA, USA). Sections were incubated with primary antibodies for 90 mins at 4°C overnight followed by 45 mins incubation with biotinylated goat anti-mouse and anti-rabbit secondary antibodies (Dako, LSAB Kit). The streptavidin peroxidase method (Dako, LSAB Kit) was performed for each staining, and the reaction was visualized with a 3-amino-9-ethylcarbazol substrate (AEC Substrate Chromogen, Dako). Sections were counterstained with Gill's No. 3 Hematoxylin (Sigma-Aldrich, MO, USA). For quantification, AEC-positive cells (red reaction product) and total nuclei (blue) per high-power field (400× magnification) were counted and expressed as AEC-positive cells/nuclei for each marker. Ten fields were quantified for each section.

The left ventricle (LV) was fixed for 48 hours in formalin (10%) and histologically processed according to Junqueira et al. (1979) [20 (link)]. The ventricles histological sections were treated with 3% hydrogen peroxide to block endogenous peroxidase activity. The samples were incubated for 12 hours with polyclonal antibody produced in rabbit anti-CD40L (Santa Cruz^® 1:50) in a humid chamber. After incubation with the primary antibody, a second incubation with biotinylated secondary antibody (Dako^® LSAB kit) was performed for 1 hour at 37 °C. In order to demonstrate the immunoreactive areas, the sections were incubated with peroxidase-conjugated complex (Dako^® LSAB kit) for 45 minutes at 37 °C and placed in chromogen solution (50 mg DAB in 50 mL PBS with 3 mL of 10% hydrogen peroxide) for 3 minutes. After counterstaining with Harris hematoxylin (Sigma^®) for 25 seconds, the blades were mounted and analyzed under an optical microscope and the fractional percentages of the immunoreactive area for LV CD40L were acquired [11 (link)].
The ventricles histological sections were stained with Picrosirius red to evaluate and quantify the collagen of cardiac tissue by means of polarized light and with HE (hematoxylin/eosin) for morphometric analysis of cardiomyocytes.