Premium
Use of High Pressure Freezing and Freeze Substitution Processing of Tissues
Author(s) -
Gattone Vincent H,
Pennington Janice,
Miller Caroline,
Molitoris Bruce,
Bacallao Robert
Publication year - 2006
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.20.5.lb2-a
Subject(s) - cryofixation , uranyl acetate , chemistry , electron microscope , frozen section procedure , sapphire , microscope , biomedical engineering , kidney , microtome , materials science , anatomy , pathology , biology , ultrastructure , optics , medicine , laser , physics , endocrinology
Electron microscopy is a widely used morphological tool in renal research, however, chemical fixatives could cause ultratructural artifacts and fixation of antigenic epitopes can hinder immunocytochemical studies. Cryofixation using high pressure freezing can preserve biological tissues which can minimize ice crystal damage and preserve antigenic epitopes. Our laboratory processed cells (MDCK cells grown on sapphire discs) and various tissues (collected using a microbiopsy needle) using the Leica EM PACT high pressure freezer. After a rat was anesthetized, tissue from the renal cortex and medulla, liver, pancreas and uterus were obtained using the Leica microbiopsy system. Another rat was anesthetized and fluorescent tagged ligands infused into the tail vein and the kidney visualized using a two photon microscope. Sequential biopsies were taken using the microbiopsy needle and high pressure frozen. These tissues could be correlated with the 2‐photon microscope images. The frozen tissues were processed using the Automated Freeze Substitution (AFS) system. The tissue was processed in 1%OsO4 in acetone with or without 0.1% uranyl acetate / 0.1% glutaraldehyde and embedded in EMBED 812 or LR Gold. MDCK cells grown on sapphire discs froze well, without obvious freezing artifacts with excellent preservation of the cellular organelles. Kidney tissues obtained with the microbiopsy needle were generally well fixed, without significant artifacts. The current availability of high pressure freezers like the Leica EM PACT, with their compact size and mobility, make good cryofixation a more available option.