Thawed cryosections

Select a suitable area for thin sections and trim the sides of the block. This can be done on the sides of a glass knife or with a diamond trimming tool. The smaller the block the more likely it is to section well. 

Cool down to cutting temperature (-90 °C to -100 °C is suitable for many samples). 

Cut sections and remove from the knife-edge with an eyelash. 

Thaw and place onto a formvar coated, carbon coated grid. 

If the block being cut contains the area of interest and the immunolabelling is satisfactory, it is time to cut thin sections for EM immunolabelling 

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Y-D. Stierhof, H. Schwarz, M. Durrenberger, W. Villiger, and E. Kellenberger, Yield of Immunolabel Compared to Resin Sections and Thawed Cryosections, in Colloidal Gold Principles, Methods and Applications, Vol. 3 (ed. M. A. Hayat), Academic Press, New York, 1991, pp. 87-95. 

Transfer thawed cryosections onto Formvar carbon-coated nickel grids. 

The alternatives to preparing and labelling thawed cryosections are the two resin embedding methods PLT and freeze-substitution. The simplest to do is the PLT method which can be completed without the necessity of expensive equipment. 

Preparation of thawed cryosections for LM 252 Immunolabelling 0.5 (xm sections for LM 253 Preparation of thawed cryosections for EM immunolabelling 253 Immunolabelling thawed cryosections 254... 

Small pieces of tissue are fixed for 1 hr in a mixture of 4% formaldehyde and 0.5% glutaraldehyde in 100 mM phosphate buffer (pH 7.4) (Chicoine and Webster, 1998). They are infiltrated with 2.3 M sucrose for 24 hr at 4°C, mounted with specimen pins (Leica, Deerfield, IL), and frozen by immersion in liquid nitrogen. The frozen tissue is sectioned at -110°C using an Ultracut E ultramicrotome (Leica) equipped with a diamond knife and an FC4 cryoattachment. Cryosections -60-80 nm thick are thawed and then mounted on Formvar-carbon-coated grids. 

Cryosectioning sections smear Specimen may not be cold enough so they thaw Select lower temperature and wait until cold enough... 

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