Pan cytokeratin ae1 ae3

Serial paraffin sections were de-paraffinized, rehydrated, and blocked in 3% BSA. They were then incubated with primary antibodies overnight at 4 °C. Antibodies used were: CK8/18 (1:200, Fitzgerald, #20R-CP004, Suffolk, England), Synaptophysin (1:100, Sigma/Cell Marque, #336R-95/clone MRQ-40, St. Louis, MO), Insulin (1:100, Cell Signaling Technology, #4590 S, Danvers, MA), Pan-Cytokeratin AE1/AE3 (1:200, Santa Cruz, #sc-81714, Dallas, TX), Nestin (1:100, Abcam #22035, Cambridge, UK). Sections were then incubated with AlexaFluor 594 or 488 secondary antibodies (1:200, Life Technologies, Grand Island, NY) and, DAPI (1:2000) and mounted using prolong gold anti-fade reagent (Life Technologies, Grand Island, NY).

Tissue samples for histological analysis were taken at the time of sacrifice from liver, spleen, skeletal muscle, heart, gonads, lung and kidney. In order to ensure that all the liver was accurately represented, samples from four liver lobes per animal were collected. Samples were fixed in 4% PFA-sucrose, embedded in paraffin and 7 µm sections were cut. Sections were stained with hematoxylin-eosin (HE) for analysis of liver histology. Histology was assessed in a blinded manner by a hepatopathologist (V.K.) from 4–5 slides/liver and changes were graded according to severity and/or prominence of each change from 0 to 3^{34 (link)}. The average score for each treatment group ± SEM is shown.
Immunohistochemical staining was done for macrophages (1:200, RAM-11; DAKO), proliferating cells (1:200, Ki-67; DakoCytomation), rabbit T-lymphocytes (1:200; GenWay), Cyr61 (5 µg/ml; Cyr61, Novus Biologicals;), β-galactosidase (1:200; β-gal; Promega) and cytokeratins (1:200, pan-Cytokeratin, AE1/AE3; Santa Cruz Biotechnology). Photomicrographs of the 7 µm histological sections were taken with an Olympus AX70 microscope (Olympus Optical) and analySIS software (Soft Imaging System). Images were further processed for publication with Adobe Photoshop 7.0 (Adobe).