Preparation of collagen gel matrices for light and electron microscopy

Cells grown on type I hydrated collagen gels require special techniques for sample preparation and processing in order to optimize the removal of all reagents from the collagen matrix and prevent artifactual shrinkage. This method includes cutting out a small block of the collagen gel, postfixation, and transfer to a scintillation vial for further processing. These additional steps ensure that all sides of the block will come in contact with solutions and reduces the possibility of reagent trapping within the collagen matrix. Additionally, in our study the collagen gel and endothelial cells that form a monolayer on the surface are oriented to allow the microtomist greater assurance of cutting true cross sections, thus saving time and increasing reproducibility. The dehydration sequence is also modified, with an increase in the times and additional steps, especially in the higher concentrations of dehydrant.