Anti cd31

FFPE tumour samples were sectioned and stained with haematoxylin and eosin (H&E), or the following antibodies: anti-pan-cytokeratin (mouse, AE1/AE3, Dako, North Sydney, NSW, Australia), anti-CD31 (mouse, JC70A, Dako), anti-CD34 (mouse, QBEnd10, Dako), anti-ERG (rabbit, EPR3864, Roche Diagnostics, North Ryde, NSW, Australia), anti-CAMTA1 (rabbit, polyclonal, Novus Biologicals, Noble Park North, VIC, Australia), and anti-TFE3 (rabbit, EPR11591, Abcam, Melbourne, VIC, Australia). H&E and IHC slides were scanned digitally at 20× magnification using the Pannoramic 1000 scanner (3DHISTECH Ltd., Budapest, Hungary). High-definition images were uploaded into CaseCenter (3DHISTECH Ltd.), and images were processed using FIJI image analysis software 2.14.0 [46 (link)].

Immunohistochemistry was performed using mouse monoclonal anti-Ki67 and anti-CD31 (Dako, Agilent Technologies, Glostrup, Denmark; available on: http://www.dako.com) [45 (link),46 (link),47 (link),48 (link)]. The percentage of Ki67⁺ cells in the basal layer of the epidermis and in the outer root sheath of hair follicles, and the number of vessels per mm² were calculated according to morphometric criteria.

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.

We used paraffin embedded frontal lobe brain sections from three deceased CADASIL patients (I: female age 59, p.Arg153Cys; II: female age 57, p.Arg153Cys; III: male age 70, p.Cys446Phe) and three deceased controls with no known cerebrovascular disorders (I: male age 67, II: male age 58, III: male age 53). Sections were de-waxed, rinsed with ethanol and blocked with methanol/H₂O₂. After heat-induced antigen retrieval in 0.01 M citrate buffer pH 6, slices were washed three times with PBS, and incubated overnight at room temperature with a 1:1 cocktail of anti-NOTCH3^ECD (dilution 1:500) and anti-CD31 (Dako, Glostrup, Denmark; dilution 1:50). The following day, sections were washed and incubated for 1 hour at room temperature with a 1:1 cocktail of anti-rabbit Envision/HRP (Dako) and goat anti-mouse alkaline phosphatase (Vector Laboratories, Burlingame, CA, USA; dilution 1:25). Finally, sections were sequentially developed with 3,3′-diaminobenzidine solution and Vector Blue (Vector laboratories). Per individual, four images were taken at a 400× magnification on a Leica IM 500 microscope and analysed using ImageJ software. The vessel area was selected manually based on a positive CD31 staining. Within the vessel area, the NOTCH3 score was calculated using an intensity threshold of 100.

MMA embedded sections were cut at 5μm thickness and stained with hematoxylin-eosin (H&E) and Movat Pentachrome. Immunoflourescent staining of endothelial cells was performed by labelling against CD31 (Dako Corp., Via Real - USA). Samples were initially incubated in 0.1% Triton X for 20 minutes and rinsed with PBS. The stent half was then subsequently exposed overnight at 4 °C to anti-CD31 (Dako Corp., Via Real – USA; dilution 1:20). The antibody reaction was visualized with an Alexa Fluor 555 donkey anti-mouse secondary antibody (Life Technologies, Carlsbad, CA dilution 1:150). DAPI (Life Technologies, Carlsbad - USA) was used as the nuclear counter stain. Selected cross-sections from rabbit iliac arteries were also stained with antibodies against RAM-11 (Dako Corp., Via Real – USA) to identify macrophages and foam cells.