Computational protocol: Intestinal epithelial cell caveolin 1 regulates fatty acid and lipoprotein cholesterol plasma levels

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Protocol publication

[…] Approximately 24 h prior to imaging, larvae were heat shocked as described above to induce Cav1-eGFP expression. Larvae were anesthetized with tricaine (Sigma-Aldrich, St Louis, MO) and mounted in 3% methyl cellulose (Sigma-Aldrich) under a coverslip (). Live Tg(hsp70:cav1-eGFP) larvae were imaged on a SP-2 confocal microscope (Leica Microsytems, Deerfield, IL) with an argon laser under a 63× oil immersion objective. Images were collected as 12 bit and analyzed with Metamorph software (Molecular Devices, Sunnyvale, CA). Regions of the brush border, lateral and basolateral PM, as well as intracellular regions, were outlined for quantification of mean fluorescence intensity (three regions of interest for each cellular area, three images per fish, three fish per experiment, for a total of three experiments or nine fish). [...] CAV1 protein was measured by western blot as previously described (). Briefly, cytosolic fractions of jejunum were run on denaturing SDS-PAGE gels with a Mini-Protean system (Bio-Rad Laboratories), transferred to nitrocellulose, blocked with blocking grade blocker (Bio-Rad Laboratories), and probed with antibodies to CAV1 (#610059, BD Transduction Labs, Lexington, KY; 1:5000 dilution), α-tubulin (#T6199, Sigma-Aldrich; 1:10,000 dilution), goat anti-rabbit IgG–HRP (#170-6515, Bio-Rad Laboratories; 1:5000 dilution), and goat anti-mouse IgG–HRP (#170-6516, Bio-Rad Laboratories; 1:10,000 dilution). Protein expression was imaged by chemiluminescence (SuperSignal West Pico Chemiluminescent Substrate, Pierce, Rockford, IL). Data from three western blots, with five WT and five CAV1IEC-KO mice each, were analyzed with ImageJ software (NIH, Bethesda, MD). […]

Pipeline specifications

Software tools MetaMorph, ImageJ
Applications Laser scanning microscopy, Microscopic phenotype analysis
Organisms Mus musculus, Danio rerio
Diseases Cardiovascular Diseases, Diabetes Mellitus, Type 2, Hypertension, Atherosclerosis, Chemical and Drug Induced Liver Injury
Chemicals Cholesterol, Fatty Acids, Nonesterified, Palmitic Acid