Plunge Frozen Grids
Plunge Frozen Grids - Related Videos
09:59
Preparation of Primary Neurons for Visualizing Neurites in a Frozen-hydrated State Using Cryo-Electron Tomography
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Cited by 24 •
2014
To preserve neuronal processes for ultrastructural analysis, we describe a protocol for plating of primary neurons on electron microscopy grids followed by flash freezing, yielding samples suspended in a layer of vitreous ice. These samples can be examined with a cryo-electron microscope to visualize structures at the nanometer scale.
02:37
Frozen Mouse Brain Tissue Sectioning: A Procedure to Obtain Thin Frozen Tissue Sections from Frozen Murine Brain Tissue
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2023
In this video, we demonstrate the sectioning of a frozen mouse brain tumor tissue using a cryostat. The frozen brain tissue sections so obtained are stored at low temperatures until further analysis.
08:01
Visualizing Proteins and Macromolecular Complexes by Negative Stain EM: from Grid Preparation to Image Acquisition
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Cited by 137 •
2011
Visualizing protein samples by negative stain electron microscopy (EM) has become a popular structural analysis method. It is useful for quantitative structural analysis, such as calculating a 3D reconstruction of the molecules being studied, and also for qualitative examination of the quality of protein preparations. In this article we present detailed protocols for preparing the EM grids, staining the sample and visualizing the sample in an electron microscope. Novice users can follow these...
07:28
Paraffin-Embedded and Frozen Sections of Drosophila Adult Muscles
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Cited by 42 •
2010
Identification of mechanisms underlying muscle damage is crucial. Here we present the histological technique for preparing paraffin-embedded and frozen sections of Drosophila thoracic muscles. This allows analysis of muscle morphology and localization of protein and other muscle cell components.
11:39
Using Unfixed, Frozen Tissues to Study Natural Mucin Distribution
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Cited by 39 •
2012
Unfixed frozen tissue samples embedded in Optimal Cutting Temperature medium (OCT) can be used to study natural distribution and glycosylation of secreted mucus. In this approach tissue processing is minimal and the natural presentation of glycolipids, mucins and glycan-epitopes is preserved. Tissue sections can be analyzed by immunohistochemistry using fluorescence or chromogenic detection.
FAQs
Vitrification keeps water from forming damaging ice crystals by cooling the applied sample extremely rapidly. In this workflow, liquid ethane serves as the cryogen and is cooled by liquid nitrogen. The resulting amorphous, glass-like water preserves biological material in a hydrated state suitable for subsequent electron microscopy.
Blotting removes excess liquid so the grid retains a thin layer of sample before freezing. That thin layer allows the material to be rapidly cooled throughout, supporting vitrification rather than crystal formation. Consequently, controlling the amount of liquid left on the grid is central to preparing a specimen that can preserve structural information.
Because the sample remains hydrated and is not chemically fixed or dehydrated, Plunge Frozen Grids can retain structural features closer to the specimen’s near-native condition. This distinguishes the preparation from workflows that alter the sample through fixation or water removal. In biochemistry, that preservation is important when interpreting proteins, complexes, or cellular materials structurally.
The workflow begins by placing the biological sample on an electron microscopy grid, removing excess liquid by blotting, and rapidly plunging the grid into liquid ethane cooled with liquid nitrogen. Each step prepares the thin specimen layer for vitrification, while the final plunge establishes the frozen hydrated state needed for imaging.
These grids support both single-particle analysis and electron cryotomography. Single-particle analysis is relevant when researchers examine individual proteins or complexes, whereas electron cryotomography extends the use of vitrified material to cellular materials. Together, these approaches make the grids useful across biochemical and structural studies that require preserved biological organization.
Plunge Frozen Grids connect biochemical sample preparation with structural biology by allowing proteins, complexes, and cellular materials to be examined after rapid freezing rather than fixation or dehydration. Their value lies in retaining hydrated, near-native structural context, which supports interpretation of molecular organization through cryo-electron microscopy methods such as single-particle analysis and electron cryotomography.