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Membrane alterations in cerebral cortex when using PIPES buffer.

R L Schultz, D O Wagner

    Journal of Neurocytology
    |August 1, 1986
    PubMed
    Summary
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    PIPES buffer in glutaraldehyde fixatives causes artifactual multivesicular myelin figures in brain tissue. Alternative buffers like cacodylate or phosphate, or adjusted fixative concentrations, prevent these artifacts, improving tissue preservation for research.

    Area of Science:

    • Neuroscience
    • Cell Biology
    • Histology

    Background:

    • Vascular perfusion fixation is crucial for preserving brain ultrastructure.
    • Artifacts can arise from fixation protocols, complicating interpretation of cellular morphology.
    • PIPES buffer is commonly used in biological buffers, but its effect on fixation artifacts requires investigation.

    Purpose of the Study:

    • To investigate the formation of artifactual structures during brain perfusion fixation using PIPES-buffered glutaraldehyde.
    • To identify factors influencing the occurrence and morphology of these artifacts.
    • To compare the effects of different buffer systems on brain tissue preservation.

    Main Methods:

    • Vascular perfusion of rat brains with 0.1 M PIPES-buffered 3% glutaraldehyde.

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  • Comparison with cacodylate- or phosphate-buffered fixatives.
  • Variations in fixative concentration and perfusion rate were tested.
  • Analysis of artifact morphology, specifically multivesicular myelin figures, in the cerebral cortex.

    • Main Results:

    • PIPES-buffered glutaraldehyde induced expanded, vesicle-filled cell processes (multivesicular myelin figures), primarily in layer 2 of the cerebral cortex.
    • These artifacts were absent when cacodylate or phosphate buffers were used.
    • Higher aldehyde concentration reduced artifact size and number.
    • Slower perfusion rates increased artifact size and frequency with PIPES buffer.

    Conclusions:

    • PIPES buffer, due to its non-toxic nature, may permit the formation of membranous artifacts during fixation.
    • Phosphate and cacodylate buffers appear to interfere with cellular activity, preventing artifact formation.
    • Optimizing buffer choice and fixation parameters is essential for accurate ultrastructural studies of the brain.