title: Assessment of human islet grafts in frozen sections of CD-1 athymic nu/nu mouse liver for molecular analysis.
abstract: BACKGROUND
  Post-transplantation islet graft monitoring is hampered by a lack of efficient methods to locate and analyze islets in situ. We evaluated histologic methods to rapidly locate islets within the liver parenchyma post-transplantation, using several staining strategies, prior to analysis using laser capture microdissection.
  
  
  METHODS
  Human islets were isolated (n = 8) from brain dead, multiorgan donor pancreases at the McGill University Health Centre Islet Transplant Laboratory. Mean yield was 247,609 ± 195,272 IE and 3172 ± 1645 IE/g (purity and viability, respectively, 84.5 ± 8.6% and 95 ± 5% average; mean ± SD). Diabetic athymic CD-1 nu/nu mice (streptozotocin intraperitoneal injection, 200 mg/kg) were maintained with sustained release insulin pellets until a suitable islet preparation was available for transplant. Intraportal islet transplantation of 2000 IE/mouse was performed via the ileocecal vein, as previously described. Frozen sections of liver containing human islets were prepared from specimens collected on days 0, 4, and 30 post-transplant. Every twentieth slide from serial sectioned liver was stained using a rapid protocol to determine if islets were present. Sections were fixed and stained for 5 minutes with either an anti-human insulin fluorescein isothiocyanate (FITC)-conjugated primary antibody (Ins-FITC), Newport Green (NG), or diphenylthiocarbazone (dithizone, DZ).
  
  
  RESULTS
  Islets were readily localized using each technique, mostly toward the liver periphery. However, DZ had a faint appearance in 10-μm-thick sections and was best utilized to locate sections containing islets during sectioning.
  
  
  CONCLUSIONS
  Ins-FITC, NG, and DZ are all good candidates for a rapid islet staining protocol to evaluate human islet grafts in situ, with DZ being best for sectioning and Ins-FITC and NG being equal in locating islets during processing for laser capture microdissection.
authors.affiliation: Human Islet Transplant Laboratory, McGill University Health Centre, Montréal, Québec, Canada; Department of Surgery, McGill University, Montréal, Québec, Canada.
authors.forename: C P
authors.initials: CP
authors.lastname: Hasilo
authors.affiliation: Human Islet Transplant Laboratory, McGill University Health Centre, Montréal, Québec, Canada; Department of Surgery, McGill University, Montréal, Québec, Canada.
authors.forename: S
authors.initials: S
authors.lastname: Negi
authors.affiliation: Human Islet Transplant Laboratory, McGill University Health Centre, Montréal, Québec, Canada.
authors.forename: M
authors.initials: M
authors.lastname: Gasparrini
authors.affiliation: Human Islet Transplant Laboratory, McGill University Health Centre, Montréal, Québec, Canada; Department of Surgery, McGill University, Montréal, Québec, Canada. Electronic address: steven.paraskevas@mcgill.ca.
authors.forename: S
authors.initials: S
authors.lastname: Paraskevas
mesh_terms: D000818:Animals; D003921:Diabetes Mellitus, Experimental / Q000209:etiology / Q000473:pathology* / Q000601:surgery*; D004230:Dithizone; D016650:Fluorescein-5-isothiocyanate; D005456:Fluorescent Dyes; D005629:Frozen Sections; D006801:Humans; D007328:Insulin; D016381:Islets of Langerhans Transplantation; D061346:Laser Capture Microdissection; D008099:Liver / Q000473:pathology*; D051379:Mice; D008819:Mice, Nude; D046228:Microarray Analysis; D013194:Staining and Labeling / Q000379:methods*; D013311:Streptozocin; D014183:Transplantation, Heterologous
chemical_list: D005456:Fluorescent Dyes; D007328:Insulin; D013311:Streptozocin; D004230:Dithizone; D016650:Fluorescein-5-isothiocyanate