Dissection of intestines from larval zebrafish for molecular analysis

Abstract

ABSTRACTEpigenetic data obtained from whole zebrafish embryos or larvae may mask or dilute organ-specific information. Fluorescence activated cell sorting can diverge cells from their native state, and cryosections often yield insufficient material for molecular analysis. Here, we present a reproducible method for larval intestinal isolation at 5, 7, and 9 days post-fertilization, using the intestine-specific transgenetgBAC(cldn15la:GFP). With tweezers, the intestine can be pulled out of the abdomen in one smooth motion. Upon removal of adhering tissues, intestines can be directly used for analyses. Each dissection takes 3-6 minutes per fish. We demonstrate that 10 and 25 dissected intestines yield enough material for RNA-sequencing and ChIP-sequencing, respectively. This method results in high quality, live material, suitable for many downstream applications.METHOD SUMMARYWe present a reproducible method for zebrafish larval intestinal isolation which results in high quality, live material. With tweezers, the intestine can be pulled out of the abdomen and after removal of adhering tissues, intestines can be directly used for analyses. We demonstrate that 10 and 25 dissected intestines yield enough material for RNA-sequencing and ChIP-sequencing, respectively.