Anti podoplanin

Immunohistochemical analysis of paraffin-embedded specimens was performed as previously described [29 (link), 37 (link), 38 (link)]. Antibodies anti-ΔNp63 (anti-p40; Calbiochem, Gibbstown, NJ, USA), anti-HβD1 (Biologo, Kronshagen, Germany), anti-HβD2 (Abcam, Cambridge, MA, USA), anti-HβD4 (Abcam), anti-CD105 (Thermo Scientific, Waltham, MA, USA), anti-Podoplanin (Clone D2-40, Dako, Glostrup, Denmark), anti-alpha Smooth muscle actin (SMA) (Abcam) and anti-Prox1 (ReliaTech GmbH, Wolfenbuettel, Germany) were used for the primary reaction. Immunoperoxidase staining was performed using the Envision kit (Dako, Glostrup, Denmark) or the BrightVision Plus kit (Immunologic, Duiven, Netherlands) according to the supplier's recommendations. Positive cells were visualized using a 3, 3'-diaminobenzidine (DAB) substrate and the sections were counterstained with hematoxylin. A control IgG was used as negative control (Santa Cruz Biotechnology, Santa Cruz, CA, USA). To test the specificity of the HβD staining, the different anti-HβD antibodies were neutralized by the incubation with an excess of peptide. No immunoreactivity was observed in this condition.

Sections were pretreated in CC1‐buffer (Cell Conditioner 1, Ventana Medical Systems, Inc, Tucson, AZ, USA) at 95°C for 36 min (E‐cadherin and β‐catenin) and at 95°C for 64 min (podoplanin and Ki‐67). Antibodies were diluted in Ventana antibody diluent and incubation performed at 36°C for 32 min. For detection, Ultra View or Opti View (for podoplanin) Universal DAB Detection kit using a Bench Mark Ultra (Ventana Medical Systems, Inc, Tucson, AZ, USA) was used. Counterstaining was performed with haematoxylin and bluing reagent (Ventana). The following antibodies were used: anti E‐cadherin (M3612, DAKO), diluted 1:25; anti‐β‐catenin (SIGMA‐Aldrich), diluted 1:1500; and anti‐podoplanin (DAKO, M3619), diluted 1:10. In order to map proliferation of the tumours, an antibody against Ki‐67 (CONFIRM, clone 3090; Ventana; Tucson, AZ, USA) ready diluted was also used.

For immunofluorescence (IF), deparaffinisation, and hydration, thermal epitope demasking was done using a low pH Target Retrieval Solution (Agilent Technologies) for 20 min at 97°C in a Dako PT Link (Dako) apparatus. Sites of non-specific binding were blocked using 3% BSA in PBS (1 h/RT). The slices were incubated at 4°C overnight with primary anti-POSTN antibodies (dilution 1:200; code no. NBP1-82472; Novus Biologicals) in 3% BSA/PBS, anti-Podoplanin (ready-to-use, clone D2-40 (PDPN), code ISO072; Dako), and anti-αSMA (ready-to-use, clone IS611, code 1A4; Dako). Next, the preparations were incubated for 1 h with donkey anti-rabbit secondary Alexa Fluor 568 conjugated antibody (dilution 1:2000, Abcam, Cambridge, UK, Cat#ab175470, RRID:AB_2783823) and anti-mouse Alexa Fluor 488 conjugated antibody (dilution 1:2000, Abcam, Cambridge, UK, Cat# ab150113, RRID:AB_2756499).
Negative controls were performed with 1% BSA in PBS instead of the specific antibody. The preparations were mounted in a Prolong DAPI Mounting Medium (Invitrogen, Carlsbad, CA, USA). The observations were made at objective 60×/1.40 oil; with the use of a Fluoview FV3000 confocal microscopy (Olympus, RRID:SCR_017015) coupled with Cell Sense software (Olympus, RRID:SCR_016238).