Fig 2: Gene expression and protein levels of CB2 receptor in DM-affected dogs. Gene expression of the CB2 receptor (CNR2) measured by qPCR (A), as well as western blot analysis for this receptor (B) in spinal cord samples of DM-affected and age-matched control dogs. Values correspond to % over control animals and are expressed as means±s.e.m. for 7 animals per group. Data were analysed using the unpaired Student's t-test (*P<0.05 compared with control animals).

Fig 3: Double immunofluorescence analysis for CB2 receptor and GFAP in spinal cord sections of DM-affected dogs. Representative photomicrographs showing double immunofluorescence analysis for the CB2 receptor and GFAP, using TOPRO-3 for labelling cell nuclei, in the grey matter of the ventral horn in T7-T10 spinal cord sections of DM-affected and age-matched control dogs (n=3/group). Scale bar: 50 µm. Arrows indicate cells labelled with the antibodies for the two markers.

Fig 4: CB2 receptor immunostaining and quantification in DM-affected dogs. Representative photomicrographs for CB2 receptor immunostaining using DAB (A) and its quantification (B) in the grey matter of the ventral horn in T7-T10 spinal cord sections of DM-affected and age-matched control dogs. Values are expressed as means±s.e.m. for 5-6 animals per group. Boxed region in DM dog image is shown enlarged in panel below. Data were analysed using the unpaired Student's t-test (*P<0.05 compared with control animals). Scale bars: 150 µm and 50 µm (enlargement). Arrows indicate CB2 receptor-positive cells.

Fig 5: Human corneal epithelial cells express endocannabinoid receptors and endocannabinoid metabolizing enzymes in vitro: (A,B) CB1-specific, (C,D) CB2-specific, (E,F) NAPE-PLD-specific, (G,H) DAGLa-specific, (I,J) DAGLß-specific, (K,L) FAAH-specific, and (M,N) MAGL-specific immunoreactivity, as shown by immunofluorescence (green fluorescence) on human corneal epithelial cell cultures. Nuclei were counterstained with DAPI (4',6-diamidino-2-phenylindole; blue fluorescence). (O) Negative control of panels C, D, G–N; (P) negative control of panels A, B, E, and F. Scale bars mark 100 µm or 50 µm, depending on magnification (200× and 400×, respectively).