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Laser capture microdissection and two-dimensional polyacrylamide gel electrophoresis: evaluation of tissue preparation and sample limitations.

Author(s): Craven RA, Totty N, Harnden P, Selby PJ, Banks RE

Publication: Am J Pathol, 2002, Vol. 160, Page 815-22

PubMed ID: 11891180 PubMed Review Paper? No

Purpose of Paper

The purpose of this paper was to determine if the type of histological stain or post-fixative, followed by subsequent immunolabeling affect protein recovery or quality from cells collected by laser cell microdissection (LCM) as determined by two-dimensional polyacrylamide gel electrophoresis (2-D PAGE).

Conclusion of Paper

Protein recovered from LCM isolated cells displayed differences in percent recovery, protein focusing, and relative intensity in a histological stain-dependent manner, with methyl green and hematoxylin and eosin yielding similar and acceptable results, and toluidine blue displaying evidence of detrimental effects. Fixation method also affected recovered protein, with ethanol out performing acetone in regards to protein yield, 2D separation and spot variability.

Studies

  1. Study Purpose

    The purpose of this study was to determine if protein recovery and quality are affected by the type of histological stain in OCT-embedded LCM isolated cell populations from normal cervix, and normal and neoplastic kidney specimens.

    Summary of Findings:

    LCM cells isolated from hematoxylin and eosin stained sections (that were post-fixed in ethanol) yielded a 60-80% protein recovery rate and reproducible protein profiles. Protein focusing was adversely affected in hematoxylin and eosin stained cells compared to unstained controls and was linked, in part, to eosin staining. While 2D-PAGE analysis of methyl green stained cells resulted in high quality protein separation, changes in relative intensity were observed yet reproducible. Results in toluidine blue stained cells were more obvious, with protein recovery, intensity, and focusing adversely affected.

    Biospecimens
    Preservative Types
    • OCT
    • Ethanol
    Diagnoses:
    • Normal
    • Neoplastic - Carcinoma
    Platform:
    AnalyteTechnology Platform
    Protein 1D/2D gels
    Protein MALDI-TOF MS
    Pre-analytical Factors:
    ClassificationPre-analytical FactorValue(s)
    1D/2D gels Specific Type of tissue stain Hematoxylin and eosin
    Methyl green
    Toluidine blue
    View more
  2. Study Purpose

    The purpose of this study was to determine if different post-fixation methods (ethanol, acetone) of OCT-embedded sections and subsequent immunostaining adversely affects protein recovery or quality in LCM cells from normal cervix, or normal and neoplastic kidney.

    Summary of Findings:

    Protein recovery and separation of immunostained LCM cells were dependent upon the method of post-fixation, with ethanol out performing acetone in terms of protein yield (40 vs. 20%), 2D-PAGE separation, and reduced variability among protein profiles. The degree of protein enrichment gained by LCM was tissue-dependent, with cervical tissue alone yielding an obvious increase via 2D gel analysis.

    Biospecimens
    Preservative Types
    • OCT
    • Other Preservative
    • Ethanol
    Diagnoses:
    • Normal
    • Neoplastic - Carcinoma
    Platform:
    AnalyteTechnology Platform
    Protein 1D/2D gels
    Protein MALDI-TOF MS
    Pre-analytical Factors:
    ClassificationPre-analytical FactorValue(s)
    Biospecimen Preservation Type of fixation/preservation Acetone
    Ethanol
    Biospecimen Acquisition Biospecimen location Kidney
    Cervix
    View more

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