Isoparaffin h

Paraffin-embedded human tissue sections were de-waxed in isoparaffin H (Panreac, Castellar del Vallès, Spain) and hydrated by quick changes through serial ethanol baths. Antigen retrieval was performed by boiling in sodium citrate buffer (10 mM sodium citrate, 0.05% Tween 20, pH 6.0, in dH₂O). Samples were subjected to several washes with dH₂O and washing buffer (NaCl 8.3 g/L, Tris 1.2 g/L; pH 7.4). Blocking was performed with 10% goat serum in PBS, for 1 h at RT. Primary antibodies were diluted in Antibody Diluent (Dako) and incubated overnight at 4 °C. The following primary antibodies were used: anti-FAAH (Abcam #ab128917, dilution 1:100), anti-CK8 [DSHB (Iowa, IA, USA) #531826, dilution 1:50], and anti-SMA (DSHB #2289065, dilution 1:50). Samples were rinsed 2 × 5’ in PBS + 0.25% triton and incubated with fluorescent secondary antibodies [Invitrogen (Waltham, MA, USA), dilution 1:200] and 1 μg/mL DAPI for 1 h at RT. Finally, they were mounted with Mowiol® mounting medium. Fluorescence confocal images were acquired by using an Olympus FV1200 microscope.

Paraffin-embedded tissue sections were de-waxed in isoparaffin H (Panreac) and hydrated by quick changes through serial ethanol baths. Antigen retrieval was performed by boiling in sodium citrate buffer (10 mM sodium citrate, 0.05% Tween 20, pH 6.0, in dH₂O). Samples were subjected to several washes with dH₂O and washing buffer (NaCl 8.3 g/L, Tris 1.2 g/L; pH 7.4). Endogenous peroxidase activity was saturated by incubation with 3% H₂O₂. Blocking, primary [FAAH (Abcam #ab128917, dilution 1:100), PCNA (EMD Millipore, clone PC10, dilution 1:10.000) and CXCR4 (Abcam #124824, dilution 1:500)] and secondary antibody incubations and immunodetection were performed with the ImmPRESS™ Polymer Detection kit (Vector) following the manufacturer’s instructions. Samples were finally counterstained with hematoxylin, dehydrated in ethanol, and mounted in Eukitt® Quick-hardening mounting medium (Sigma-Aldrich).