Protein block solution

Triplicate cores were made of 118 endometrial tumors at our institution from hysterectomy specimens, and tissue microarrays were created. Immunohistochemical analysis DRD2 (1:300, Santa Cruze Biotechnology Inc.) was performed on 4-μmol/L sections of formalin-fixed, paraffin-embedded tissues using standard methodologies in UNC-CH Translational Pathology Laboratory Core. Individual slides were scanned using the Aperio™ ScanScope (Aperio Technologies, Vista, CA), and digital images were analyzed using Aperio™ ImageScope. Non-parametric tests and Cox regression analysis were used to correlate DRD2 expression with clinical outcomes.
The mouse endometrial tumor tissues were formalin-fixed and paraffin-embedded at the Animal Histopathology Core Facility at UNC-CH. Slides (5 μm) were first incubated with protein block solution (Dako) for 1 h and then with the primary antibodies for Ki-67 (1:400), phosphorylated-S6 (1:300), VEGF (1:800) and BCL-2 (1: 1200) for 2 h at room temperature. The slides were then washed and incubated with appropriate secondary antibodies at room temperature for 1 h. Further processing was carried out using ABC-Staining Kits (Vector Labs, Burlingame, CA) and hematoxylin. Immunohistochemistry slides were scanned by Motic (Houston, TX) and scored by ImagePro software (Vista, CA).

Immunohistochemical detection of beta-catenin, and PrKD1 was performed on mouse prostate tissue and transfected and non-transfected cell lines using anti-beta-catenin (Santa Cruz, #SC1496) and anti PKC mu (Santa Cruz, # 693)⁸. Mouse prostate tissue was fixed in 4% paraformaldehyde. The paraffin-embedded paraformaldehyde-fixed mice tissue was cut at 5 micron thickness. The first slice of staining sections was used for histological confirmation by Hematoxylin and Eosin (H&E) staining. The tissue slides and fixed cells were treated with 0.1% Triton-X-100 for 3 min, followed by blocking nonspecific binding using protein block solution (Dako, #X0909), overnight incubation with primary antibody at 4°C and one hour incubation with fluorescent secondary antibodies AF594 (Life technologies #SA5-10088) or AF488 (life technologies, #A11070) and contra-stained with DAPI for 5 minutes. Goat isotype IgG (Santa Cruz) was used as negative controls. Tissue slides were examined using Olympus Fluoview (FV10i) confocal microscope and cells were analyzed by IN Cell analyzer 2000 cell imaging system (GE Healthcare Life Sciences).

First, tissue sections were fixed in formalin for 12 h, embedded in paraffin, deparaffinized in xylene, and rehydrated in alcohol. Second, tissue sections were incubated 45 min in a steamer using protein retrieval solution (Dako, Glostrup, Denmark), followed by 3% hydrogen peroxide solution for 10 min and protein block solution (Dako) for 20 min at room temperature. Tissue sections were then incubated overnight with antibodies against Ki-67 (9027T, Cell Signaling Technology, Danvers, MA, USA) and AKT (4691S, Cell Signaling Technology) at 4 °C followed by 30 min with the biotinylated secondary antibody (Vector Laboratories, Burlington, CA, USA), and ABC reagent (Vector Laboratories) for 30 min. Finally, 3,3′-diaminobenzidene (DAB; Dako) was used to visualize the target proteins. Images of stained tissue sections were obtained using a IX73 microscope (Olympus, Tokyo, Japan).

To examine hepatic morphology and assess liver fibrosis, H&E staining and Sirius red staining were performed, respectively. Liver specimens were fixed in 10% neutral buffered formalin (Sigma), embedded in paraffin and cut into 4 μm sections. Next, the specimens were deparaffinized, hydrated and stained by standard methods.
For immunohistochemistry, liver sections were deparaffinized, hydrated and incubated in 3% hydrogen peroxide, to block endogenous peroxidase. Antigen retrieval was performed by heating in 10 mM sodium citrate buffer (pH 6.0) for 10 min using microwave. Specimens were blocked in Protein Block solution (Dako) for 30 min at room temperature (RT) followed by incubation with primary antibody at 4 °C overnight. Other sections were also incubated at 4 °C overnight in non-immune sera. Rabbit α-SMA antibody (diluted 1:500; Abcam) was used as a primary antibody and diluted in Protein Diluent (Dako). Polymer-horseradish peroxidase anti-rabbit (Dako) was used as secondary antibody and 3,3′-diaminobenzidine as brown colour was used to visualize the protein.

For immunohistochemistry staining, heart cryosections were fixed with 4% paraformaldehyde, permeabilized and blocked with Protein Block Solution (DAKO, Carpinteria, CA) containing 0.1% saponin (Sigma, St Louis, MO), and then incubated with the following antibodies overnight at 4 °C: mouse anti-alpha sarcomeric actin (1:100, a7811, Sigma), rabbit anti-CD45 (1:100, ab10559, Abcam, Cambridge, United Kingdom), mouse anti-Actin, α-Smooth Muscle antibody (1:100, A5228, Sigma), rabbit anti-Ki67 (1:100, ab15580, Abcam), rabbit anti-CD3 (1:100, ab16669, Abcam) and mouse anti-CD8 alpha (1:100, mca48r, abd Serotec, Raleigh, NC ). FITC- or Texas-Red secondary antibodies (1:100) were obtained from Abcam Company and used for the conjunction with these primary antibodies. For assessment of cell apoptosis, heart cryosections were incubated with TUNEL solution (Roche Diagnostics GmbH, Mannheim, Germany) and counter-stained with DAPI (Life Technology, NY, USA). For assessment of angiogram, heart cryosections were incubated with Lectin (FL-1171, Vector laboratories, Burlingame, CA, USA). Images were taken by an Olympus epi-fluorescence microscopy system.

All animals were killed 8 days after treatment. Mouse hearts were harvested and frozen in Optimal Cutting Temperature (OCT) compound (Tissue-Tek, Torrance, CA, USA). Cryo-sections (5 μm thick) were prepared. Masson’s trichrome staining was performed as per manufacturer’s instructions [HT15 Trichrome Staining (Masson) Kit; Sigma-Aldrich]. Fibrosis area was measured by NIH Image J as previously described 23 (link). For immunofluorescence staining, mouse heart cryosections were fixed with 4% PFA, blocked/permeabilized with Protein Block Solution (DAKO) containing 1% saponin (Sigma-Aldrich), and then stained with anti-α-SA (Sigma-Aldrich) and anti-von Willebrand factor (Abcam) antibodies. FITC or Texas-Red secondary antibodies were obtained from Abcam and used in conjunction with these primary antibodies. Images were taken by a Zeiss (Jena, Germany) confocal microscopy system.