Two paraffin sections of jejunum of 5 µm per mice were stained with primary antibody against perilipin-3 at dilution 1:500 (catalog abs482, Millipore, Darmstadt, Germany) followed by secondary antibody donkey anti-rabbit Dylight 594 at dilution 1:1000 (SA5-10040, Thermofisher, Waltham, MA, USA). Nuclear counterstaining was performed with Hoechst 33342. Images were captured by a Pannoramic 250 Flash III (3Dhistech, Budapest, Hungary). Percentage of staining was calculated with Visiopharm software version 6.6.3.
For hepatic lipid staining, frozen liver sections were sliced at 5 µm, treated with oil red O and scanned as previously described [27 (link)]. The lipid area was determined on whole sections using the imaging software TissueIA (version 2.0.3, Leica Biosystems, Dublin, Ireland). Pixels corresponding to the oil red O staining were selected to create a color profile. Total tissue area was defined by setting the tissue intensity threshold at 210 (grey value). Results were expressed as stained area (below threshold)/tissue area (below threshold). Two representative tissue pieces were analyzed for each mouse.
For hepatic lipid staining, frozen liver sections were sliced at 5 µm, treated with oil red O and scanned as previously described [27 (link)]. The lipid area was determined on whole sections using the imaging software TissueIA (version 2.0.3, Leica Biosystems, Dublin, Ireland). Pixels corresponding to the oil red O staining were selected to create a color profile. Total tissue area was defined by setting the tissue intensity threshold at 210 (grey value). Results were expressed as stained area (below threshold)/tissue area (below threshold). Two representative tissue pieces were analyzed for each mouse.
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