Clone jc70a

After sectioning of 4 µm slides from the formalin-fixed, paraffin-embedded blocks, deparaffinization in xylene and rehydration in a series of decreasing concentration of ethanol were done. Antigen retrieval using either the Bond Epitope Retrieval Solution 1 (pH 6) or the Bond Epitope Retrieval Solution 2 (pH 9) (Leica Microsystems, Wetzlar, Germany) at 99-100°C for 20–30 min was performed. The slides were treated with S-100 (1 : 300, clone S-100, DAKO, Carpinteria, California, USA, incubation time: 30 min in room temperature), vimentin (prediluted, clone V91, DAKO, incubation time: 30 min in room temperature), CD31 (1 : 30, clone JC70A, DAKO, incubation time: 30 min in room temperature), MDM2 (1 : 200, clone IB10, Novocastra, Wetzlar, Germany, incubation time: 45 min in room temperature), and p16 (1 : 10, clone G175-405, DAKO, incubation time: 30 min in room temperature), separately. Immunostaining was performed on Leica BOND-MAX autostainer (Leica Microsystems) and we used peroxidase/DAB Bond Polymer Refine Detection System (Leica Microsystems) for visualization. Cytoplasmic staining for vimentin and CD31 and nuclear staining for MDM2 are regarded as positive stain whereas, for S-100 and p16, both nuclear and cytoplasmic stains are required.

Immunohistochemical stainings were performed using DAKO autostainer system and commercially available endothelial markers CD31 (monoclonal mouse anti-human platelet endothelial cell adhesion molecule- (PECAM-) 1 antibody, Clone JC70A, DAKO, Glostrup, Denmark; dilution: 1 : 50 for 1 hour) and CD105 (monoclonal mouse anti-human endoglin antibody, Clone SN6h, DAKO, Glostrup, Denmark; dilution: 1 : 100 for 1 hour). As positive control, a sample of breast cancer with a known high MVD was used.

Tissue samples derived from PAR₂ induced mammary glands were fixed with 4% formaldehyde in PBS, embedded in paraffin, and sectioned (5-μm sections). After deparaffinization and rehydration, the sections were stained with H&E or subjected to immunohistochemistry. For this, the slides were incubated 3% H₂O₂ prior to antigen retrieval. Antigen unmasking was carried out heating (20 min) in a microwave oven in 10mM Tris buffer containing 1mM EDTA. After blocking slides were incubated with the following primary antibodies: anti β-catenin (C-2206, Sigma-Aldrich St Louis MO, USA), anti PCNA (sc-56, Santa Cruz Biotechnology, USA dilution 1:200) anti DVL1 (sc7397, Santa Cruz Biotechnology, Dallas Texas, USA; goat polyclonal IgG) or anti CD31 (Dako, Clone JC70A, Carpinteria, CA). Color was developed using the 3,3′-diaminobenzidine (DAB) (Thermo Scientific, Walham, MA, USA) or the Zymed AEC substrate kit (Zymed Laboratories So, San-Francisco, CA, USA), followed by counter staining with Mayer's haematoxylin. Controls without addition of primary antibodies showed low or no background staining in all cases.

The cells were washed with PBS and the aggregates were singularized using 0.25% (w/v) trypsin-EDTA (Thermo Fisher Scientific). trypsin-EDTA was neutralized using RPMI/20% (v/v) FBS medium. The cells were fixed using 2% (v/v) PFA for 20 min at room temperature. Then, the cells were centrifuged and resuspended in 90% (v/v) cold methanol, incubated for 15 min at 4 °C. The samples were then washed three times using a solution of 0.5% (v/v) BSA in PBS (FB1). Monoclonal mouse IgG primary antibody against cardiac troponin T (cTnT) (Thermo Fisher Scientific, Clone 13-1) or monoclonal mouse IgG primary antibody against CD31 (Dako, Clone JC70A, Nowy Sącz, Poland) were diluted in FB1 plus 0.1% (v/v) Triton (FB2) at 1:250 and 1:25, respectively, and incubated for 1 h at room temperature. The cells were then washed with FB2 and cell pellet resuspended with goat anti-mouse alexa-488 secondary antibody (Thermo Fisher Scientific) diluted 1:1000 in FB2 and incubated for 30 min in the dark. The cells were washed twice, and the cell pellets were resuspended in 500 µL of PBS and analyzed in a FACSCalibur flow cytometer (Becton Dickinson, Franklin Lakes, NJ, USA). The data were analyzed using FlowJo (v10, FlowJo LLC, Becton Dickinson, Franklin Lakes, NJ, USA).

The implants were removed, fixed in 4% formaldehyde, embedded in paraffin, and stained as described in [8 (link)], using anti-human HLA-A (abcam ab52922, 1:100), anti-insulin (Sigma, clone K36 AC10, 1:75), and anti-human CD31 (Dako Cytomation, Clone JC70A, M0823, 1:50 dilution) antibodies. Analysis of HLA-positive cells was done by counting cells in random five × 40 fields from each implant. Histological analysis of microvessel density (MVD) was performed as described previously [38 (link)]; microvessel density was assessed by counting CD31 positive red blood cell–filled lumens. Values reported for each experimental condition correspond to the average MVD value ± standard error of the mean (SEM) obtained from all the individual implants.
Immunofluorescence: antibodies used in this study included guinea pig anti-insulin (1:400; Dako) and mouse anti-glucagon (1:800; Abcam). Fluorescent images were taken on a Nikon C1 confocal microscope at an original magnification × 40 as described [45 (link)].