Development of rapid staining protocols for laser-capture microdissection of brain vessels from human and rat coupled to gene expression analyses

Mojsilovic-Petrovic, Jelena; Nesic, Momir; Pen, Ally; Zhang, Wandong; Stanimirovic, Danica

doi:10.1016/j.jneumeth.2003.09.026

Cited by 67 publications

(67 citation statements)

References 38 publications

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“…Using this strategy, more than 100 individual CD38+ cells were rapidly isolated from sections of an SSPE brain and analyzed for individually expressed IgG antibodies (Burgoon et al, PNAS, in press). Immunostaining protocols before LCM must be conducted quickly to preserve RNA (Fend et al, 1999;Burgess and McParland, 2002;Mojsilovic-Petrovic et al, 2004). However, since LCM was first described, few studies have examined RNA expression in individual cells from human post-mortem CNS (Bahn et al, 2001;Lu et al, 2004), likely reflecting the challenge of isolating single cells from a complex tissue and the subsequent purification of 10-20 pg of total RNA from each cell for analysis (Michel et al, 2003;Parlato et al, 2002).…”

Section: Discussionmentioning

confidence: 99%

“…Earlier reports have suggested that staining times in aqueous media should be limited to less than 20 min (Fend et al, 1999;Mojsilovic-Petrovic et al, 2004). The staining protocol used here is completed in less than 25 min and the total time required from tissue sectioning until cDNA production is less than 4 h. Careful RNase-free conditions throughout the entire procedure, conducting immunostaining on ice, and the inclusion of RNase inhibitor at every step are logical precautions to preserve RNA.…”

Section: Discussionmentioning

confidence: 99%

“…For example, immunohistochemical staining before LCM identified targeted cell types when morphological criteria failed (Fend et al, 1999;Ball et al, 2002;Vincent et al, 2002). The study of smaller numbers of cells often required preamplification of RNA from LCM-captured cells (Goldsworthy et al, 1999;Bonaventure et al, 2002;Mikulowska-Mennis et al, 2002;Ginsberg and Che, 2004) and rapid staining to preserve nucleic acid integrity in order to generate accurate gene expression profiles (Fend et al, 1999;Mojsilovic-Petrovic et al, 2004).…”

Section: Introductionmentioning

confidence: 99%

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Laser capture microdissection and single-cell RT-PCR without RNA purification

Keays

Owens

Ritchie

et al. 2005

Journal of Immunological Methods

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Chronic infectious diseases of the central nervous system (CNS) are characterized by intrathecal synthesis of increased amounts of immunoglobulin G (IgG) directed against the agent that causes disease. In other inflammatory CNS diseases such as multiple sclerosis and CNS sarcoid, the targets of the humoral immune response are uncertain. To identify the IgGs expressed by individual CD38+ plasma cells seen in human brain sections, we merged the techniques of laser capture microdissection (LCM) and single-cell RT-PCR. Frozen brain sections from a patient who died of subacute sclerosing panencephalitis (SSPE), were rapidly immunostained and examined by LCM to dissect individual CD38+ cells. After cell lysis, we developed two techniques for reversetranscription (RT) of unpurified total RNA in the cell lysates. The first method performed repeated and rapid freeze-thawing, followed by centrifugation of the cell lysate into tubes for subsequent RT. The second, more successful method performed RT in situ on detergent-solubilized cells directly on the cap surface; subsequent nested PCR identified heavy and light chain sequences expressed by two-thirds of individually isolated plasma cells. These techniques will streamline the identification of gene expression products in single cells from complex tissues and have the potential to identify IgGs expressed in the CNS of inflammatory diseases of unknown etiology.

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Laser capture microdissection and single-cell RT-PCR without RNA purification

Keays

Owens

Ritchie

et al. 2005

Journal of Immunological Methods

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“…Fresh frozen samples were collected as described above, cut into 7-m sections onto Superfrost Plus slides (MenzelGlaser), and stained in a similar fashion to a previously described protocol (46). Briefly, these were quickly fixed in 70% (v/v) ethanol, washed in PBS, stained with a fluorescent lectin (fluorescein Griffonia (Bandeiraea) simplicifolia lectin; 1/20 dilution in PBS; Vector Laboratories) for 2 min in the dark before washing again in PBS (5 ϫ 3 s).…”

Section: Laser Capture Microdissection (Lcm)mentioning

confidence: 99%

Chemokine Gene Expression during Fatal Murine Cerebral Malaria and Protection Due to CXCR3 Deficiency

Miu

Mitchell

Müller

et al. 2008

The Journal of Immunology

139

170

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Cerebral malaria (CM) can be a fatal manifestation of Plasmodium falciparum infection. Using murine models of malaria, we found much greater up-regulation of a number of chemokine mRNAs, including those for CXCR3 and its ligands, in the brain during fatal murine CM (FMCM) than in a model of non-CM. Expression of CXCL9 and CXCL10 RNA was localized predominantly to the cerebral microvessels and in adjacent glial cells, while expression of CCL5 was restricted mainly to infiltrating lymphocytes. The majority of mice deficient in CXCR3 were found to be protected from FMCM, and this protection was associated with a reduction in the number of CD8+ T cells in brain vessels as well as reduced expression of perforin and FasL mRNA. Adoptive transfer of CD8+ cells from C57BL/6 mice with FMCM abrogated this protection in CXCR3−/− mice. Moreover, there were decreased mRNA levels for the proinflammatory cytokines IFN-γ and lymphotoxin-α in the brains of mice protected from FMCM. These data suggest a role for CXCR3 in the pathogenesis of FMCM through the recruitment and activation of pathogenic CD8+ T cells.

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“…This technique has been devised to isolate distinct cell populations from their natural environment in situ not only according to morphology but also to immunophenotype criteria. [21][22][23] It has been used successfully for PCR-based gene analysis of specific cell populations in brain tissue, [24][25][26] brain vessels 27 and epithelial cells, 28,29 but to our knowledge has never been employed for genome-wide discovery. A major limitation has been the necessity to procure high quality and sufficient quantity of RNA.…”

mentioning

confidence: 99%