Immunofluorescence and Immunohistochemistry of Liver

Livers were fixed in 4% paraformaldehyde (Polysciences, Inc., Warrington, PA) and embedded in paraffin or Tissue-Tek® Optimum Cutting Temperature (OCT) Sakura Finetek, Torrance, CA) for sectioning. Immunofluorescence (IF) staining signals for PROM1 (1:50 dilution, eBiosciences, San Diego, CA), CYTOKERATIN-19 (1:100 dilution, gift from JR Friedman, University of Pennsylvania, Philadelphia. PA), and LacZ (1:400, Bioss, Woburn, MA) were detected by secondary antibodies conjugated either with anti-mouse cyanine (Cy) 3, Cy5, anti-rat Cy3/Cy5, or anti-rabbit Cy3/Cy5 (1:200 dilution, Jackson ImmunoResearch Labs, West Grove, PA) (9 (link), 10 (link), 20 (link)). Images were then acquired by a Leica DM5500B IF microscope using Leica Suite Advanced Fluorescence (LAS AF) 6000 software (Leica Microsystems, Wetzlar, Germany). Immunohistochemistry was performed using 2.1 μg/mL anti-INTEGRIN-β6 (gift from Shelia Violette, Biogen, Inc). Bright-field images were captured using a Leica DM1000 (DFC290) transmitted light microscope (Leica Microsystems AG, Heerbrugg, Switzerland) with Leica Application Suite (version 2.7.1R1).

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Zagory J.A., Fenlon M., Dietz W., Zhao M., Nguyen M.V., Trinh P., Adoumie M., Park A., Xu J., Mahdi E., Glazier A., Malkoff N., Mavila N, & Wang K.S. (2019). PROMININ-1 promotes biliary fibrosis associated with biliary atresia. Hepatology (Baltimore, Md.), 69(6), 2586-2597.

Publication 2019

paraformaldehyde Tissue-Tek® Optimum Cutting Temperature (OCT) PROM1 anti-rat Cy3/Cy5 anti-rabbit Cy3/Cy5 Leica DM5500B IF microscope

Antibodies Cytokeratin 19 Dilution Embedded paraffin Fluorescence Immunofluorescence Immunofluorescence microscope Immunohistochemistry Integrin Lacz Light microscope Livers Mouse Paraformaldehyde Rabbit Tissue

Corresponding Organization : Children's Hospital of Los Angeles

Other organizations : Cedars-Sinai Medical Center

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Variable analysis

independent variables

None explicitly mentioned

dependent variables

Immunofluorescence (IF) staining signals for PROM1, CYTOKERATIN-19, and LacZ

control variables

Liver tissue fixed in 4% paraformaldehyde and embedded in paraffin or Tissue-Tek® Optimum Cutting Temperature (OCT) for sectioning
Secondary antibodies conjugated with anti-mouse cyanine (Cy) 3, Cy5, anti-rat Cy3/Cy5, or anti-rabbit Cy3/Cy5 used for detection
Microscopy techniques used: Leica DM5500B IF microscope and Leica DM1000 (DFC290) transmitted light microscope

controls

Positive control: Not explicitly mentioned
Negative control: Not explicitly mentioned

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