3. Image Analysis

Live Staining of CLF Uptake into Bile Canaliculi

Introduction

Corning® Cholyl-lysyl-fluorescein (CLF) staining is used to visualize the structure and function of bile canaliculi in polarized hepatocytes. CLF is a substrate for the canalicular bile salt export pump (BSEP), thus it can be used to visualize BSEP-mediated canalicular efflux, as well as to label the bile canaliculi structures.


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Fluorescence Imaging

Introduction

All fluorescent or confocal imaging of cells can be done directly in the Organ-Chip. You do not need to disassemble the chip, or isolate the membrane in order to image cells. The membrane is located 0.8 mm from the bottom of the chip and is visually accessible by most objectives on most microscopes, although the use of a long-working distance objective lens is recommended for optimal results. The chip is made of an optically transparent material that will not cause any significant distortion of your signal or image; it does not auto fluoresce.


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Live Staining of CDFDA Uptake into Bile Canaliculi

Introduction

Carboxy-DCFDA (5-(and-6)-Carboxy-2',7'-Dichlorofluorescein Diacetate) is a reagent that passively diffuses into cells. It is colorless / nonfluorescdent and is cleaved by intracellular esterases to yield a fluoresecent fluirophore, 5-(and-6)-carboxy-2',7' –dichlorofluorescein (CDFDA). This reagent can be used to visualize the formation of bile canaliculi in polarized hepatocytes which is indicative of MRP-2 trasnporter activity. Carboxy-DCFDA is a substrate of multidrug resistance-associated protein 2 (MRP2), a hepatocyte efflux transporter, thus it can be used to visualize MRP2-mediated canalicular uptake and efflux, as well as to label the bile canaliculi structures.


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Immunofluorescence Staining of Stabilin-1

Introduction

This gene encodes a large, transmembrane receptor protein and is primarily expressed on sinusoidal endothelial cells of liver, spleen, and lymph node. The receptor has been shown to endocytose ligands such as low density lipoprotein, Gram-positive and Gram-negative bacteria, and advanced glycosylation end products.


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