Cells were fixed in 1% paraformaldehyde (v/v) for 25 minutes, and then washed in PBS. Ceramide labelling was conducted using an antibody from Alexis (San Diego, CA. USA). Cells that were stained for UGT8 (antibody was obtained from Abcam, Cambridge, MA. USA) were permeabilized and then the antigenic sites were un-masked using sodium citrate buffer. Immunostaining was then done using a Histostain-plus kit (Invitrogen, Carlsbad, CA) following the manufacturer’s instructions, previously described [3 (link), 6 (link)]. Images were acquired using LEICA DM LB light microscope and Leica IM1000 software.
Tumour tissues were sliced into two halves. One half was embedded in optimal cutting temperature (OCT) media, and stored at -80 C. Cryo- 8μm sections were prepared, and used for ceramide labelling as described above. The other half was fixed in freshly prepared 1% paraformaldehyde (v/v). Histopathology was assessed using hematoxylin and eosin (H&E) staining.
Tumour cell death was evaluated using a TUNEL assay and fibrosis was revealed using Masson trichrome staining. TUNEL assay results were evaluated using Image J as was previously described [6 (link), 8 (link)], where areas of cell death were quantified relative to the total area of tumour sections.
Tumour tissues were sliced into two halves. One half was embedded in optimal cutting temperature (OCT) media, and stored at -80 C. Cryo- 8μm sections were prepared, and used for ceramide labelling as described above. The other half was fixed in freshly prepared 1% paraformaldehyde (v/v). Histopathology was assessed using hematoxylin and eosin (H&E) staining.
Tumour cell death was evaluated using a TUNEL assay and fibrosis was revealed using Masson trichrome staining. TUNEL assay results were evaluated using Image J as was previously described [6 (link), 8 (link)], where areas of cell death were quantified relative to the total area of tumour sections.
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