Larval (6 dpf) zebrafish and intestines from adult zebrafish and were collected for EM. Prior to euthanasia adult fish were fed on a lipid-rich, hard-boiled chicken egg yolk for 1 h ad lib; larvae studied were lecithotrophic (6 dpf) and thus were not provided exogenous food. EM samples were fixed in 3% glutaraldehyde (Electron Microscopy Sciences, Hatfield, PA) and 1% formaldehyde, post-fixed in reduced osmium (Electron Microscopy Sciences), stained with uranyl acetate (Fisher Scientific), embedded in Epon 812 resin (Ladd Research Industries, Williston, VT), and imaged on a Technai-12 electron microscope (FEI, Hillsboro, OR) with a 794 multiscan camera (Gatan, Pleasanton, CA).
Mouse CAV1 IF was attempted on paraffin sections and cryosections of jejunum collected after a 4 h fast, but non-specific fluorescence was observed in enterocytes, even in the negative control, global CAV1 KO mice. The antibodies tested at a range of dilutions were: BD Biosciences/Transduction Labs #610059, 610057 and 610406, Santa Cruz Biotechnology #sc-894, Abcam #ab2910, and Cell Signaling #3238 s. The antigen retrieval methods tested were Tris-EDTA buffer, sodium citrate buffer, and Diva Decloaker (Biocare Medical, Concord, CA).
Mouse CAV1 IF was attempted on paraffin sections and cryosections of jejunum collected after a 4 h fast, but non-specific fluorescence was observed in enterocytes, even in the negative control, global CAV1 KO mice. The antibodies tested at a range of dilutions were: BD Biosciences/Transduction Labs #610059, 610057 and 610406, Santa Cruz Biotechnology #sc-894, Abcam #ab2910, and Cell Signaling #3238 s. The antigen retrieval methods tested were Tris-EDTA buffer, sodium citrate buffer, and Diva Decloaker (Biocare Medical, Concord, CA).