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Laser Capture Microdissection

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Science  08 Nov 1996:
Vol. 274, Issue 5289, pp. 998-1001
DOI: 10.1126/science.274.5289.998

Figures

  • Fig. 1.

    (A) Transparent EVA thermoplastic film is applied to the surface of a routine tissue section mounted on a glass slide. The tissue-EVA film sandwich is viewed under a microscope, and the cells of interest are positioned in the center of the field. (B) A focused laser beam coaxial with the microscope optics is pulsed to activate the film, causing it to become focally adhesive and fuse to the selected underlying cells in the tissue section. (C) When the EVA film is removed from the tissue section, the selected cells remain adherent to the film surface. The film is then placed directly into the DNA, RNA, or enzyme buffer. The cellular material detaches from the film and is ready for standard processing.

    LAMOST
  • Fig. 2.

    Examples of LCM transfer. Sections were dehydrated, mounted on a glass slide, and stained with hematoxylin and eosin (H/E) or silver. (A to D) LCM transfer of kidney glomeruli. Several glomeruli were removed from the kidney cortex [(A), arrows] and were transferred to film [(B), H/E stain]; scale bar, 200 μm. The size and shape of the transfer region can be adjusted by altering the pulse characteristics of the laser and the number of laser pulses used to procure a lesion. The procured glomeruli in (B) vary in size from 60 to 150 μm. The same transfer is shown in (C) and (D) for a single glomerulus (scale bars, 50 μm); note the retained histology of the transferred tissue in (D). (E to G) LCM transfer of Alzheimer's plaques in brain. Multiple lesions were procured from a section of frontal cortex [(E), arrow] and were transferred to film [(F), silver stain]; scale bar, 100 μm. A transferred lesion is shown in (G) (scale bar, 50 μm); note the darkly stained neurofibrillary tangle within the transferred tissue. (H and I) LCM transfer of in situ breast carcinoma, showing multiple regions of transfer. The lesion in the center of the field contains a region of central necrosis surrounded by viable neoplastic cells. LCM procurement (arrow) avoids both necrotic tissue and surrounding stroma (H/E stain; scale bar, 100 μm). (J and K) LCM transfer of ADH of the breast. ADH is the putative earliest precursor lesion of breast neoplasia and can occur several decades before the development of cancer. However, because of the small size of the lesion, little is known of the genetic alterations present in ADH. Note the incipient cribriforming pattern evident in the transferred lesion (circled area), a characteristic histopathological feature of ADH (H/E stain; scale bar, 100 μm). (L and M) LCM transfer of PIN, a precursor lesion of prostate cancer. The transferred PIN foci are indicated by arrows (H/E stain; scale bar, 200 μm). (N and O) LCM transfer of lymphoid germinal centers (arrows); the transferred regions are rich in B lymphocytes and are surrounded by the mantle zone, which was excluded (H/E stain; scale bar, 200 μm).

  • Fig. 3.

    Examples of PCR, RT-PCR, and enzyme activity assay after LCM transfer. (A) LCM transfer, PCR amplification, and denaturing gel electrophoresis of DNA recovered from an invasive breast cancer sample. PCR primer sets were specific for microsatellite D8S136 located on chromosome 8p21. The two lanes labeled C contained separate regions of transfer film that were placed on the tissue sample but were not laser-activated. No amplification product is visualized, indicating that nonspecific transfer of cells to the film did not occur. Lanes labeled N and T represent PCR amplification of normal and tumor DNA, respectively, showing the presence of two alleles (arrows). (B) LCM transfer of prostate cancer sample showing allelic deletion. DNA from normal epithelium (N) and tumor (T) were PCR-amplified at marker D8S339 on chromosome 8p and analyzed by denaturing gel electrophoresis. The upper allele in the tumor sample is deleted (arrow). Note the complete loss of the allele due to the purity of the procured tumor sample. (C) LCM transfer of prostate cancer sample analyzed at marker D8S339. Both the normal (N) and tumor (T) samples show the presence of one allele, indicating the patient is homozygous at this marker. (D) LCM transfer of prostate cancer sample showing allelic deletion. DNA from normal epithelium (N) and tumor (T) were PCR-amplified at marker D8S136. The lower allele in the tumor sample is deleted (arrow). (E) LCM and analysis of a gastrinoma showing LOH on chromosome 11q13 (marker D11S449) at the region of the putative MEN1 tumor suppressor gene. The upper allele in the tumor sample is deleted (arrow). (F) LCM transfer and analysis of DNA from a breast cancer sample from a patient with BRCA1 mutation-positive familial breast cancer. The tumor sample shows deletion of the upper allele (arrow) at marker D8S855 located within the BRCA1 gene on chromosome 17q21. (G) LCM transfer and analysis of an invasive squamous cell esophageal tumor. The tumor shows allelic loss in the tumor sample (arrow) at marker D9S171 in the vicinity of the p16 gene on chromosome 9p. The normal and tumor tissue samples were fixed in ethanol before paraffin embedding. (H) LCM transfer, PCR amplification, and native gel electrophoresis analysis (SSCP) of DNA from a hemangioblastoma, using PCR primers specific for exon 2 of the VHL gene on chromosome 3p25. A single-base pair mutation is present in the tumor sample (T) and appears as an additional band. The normal control tissue (N) shows two bands representing the sense and antisense DNA strands. (I) LCM transfer, RT-PCR amplification, and denaturing gel electrophoresis of actin mRNA from a frozen section of prostate cancer (+ lanes, RT-PCR-amplified mRNA from normal epithelium, tumor, and control RNA, respectively; - lanes, matching controls prepared without reverse transcriptase). (J) LCM transfer and analysis of PSA mRNA from a frozen section of prostate cancer (+ lanes, RT-PCR-amplified mRNA from prostate tumor with 1× and 10× concentrations of template, respectively; - lanes, matching controls prepared without reverse transcriptase). (K) LCM transfer, PCR amplification, and gel electrophoresis of M. tuberculosis DNA from a granulomatous lesion in lung (- lane, DNA from normal lung adjacent to the lesion; + lane, DNA from a granuloma with histological documentation of acid-fast bacilli). (L) LCM transfer of frozen prostate cancer sample analyzed by gelatin zymography. Enzymatic clearing of the gel by MMP-2 (gelatinase A) results in the presence of a single band.

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