Lab Techniques

List of Techniques

Our laboratory manager, technician, postdocs, visiting scientists, and collaborators are specialists in laboratory techniques pertinent to dermatopathology. We assist via collaborations through our basic research core and clinical research core. Collectively, we have many decades of experience with the following techniques:

 

Conventional Morphology

  • Paraffin embedding, microtomy and slide preparation
  • Histochemical staining
  • Photomicrography
  • Computer-assisted image analysis
  • Microdensitometry
  • Planar morphometry

 

Specialized Morphology

  • Confocal microscopy
  • Transmission electron microscopy
  • Immunoelectron microscopy (including multiple epitope detection with colloidal gold)
  • Tissue microarray quantitation (Chromovision system)

 

 

In Vitro/In Vivo Modeling Systems

  • Skin organ culture
  • Tissue culture
  • Skin three-dimensional reconstructs
  • Humanized SCID mouse xenografts
  • Negative pressure chamber models

 

 

Tissue Protein Antigen Detection

  • Cryomicrotomy
  • Immunohistochemistry (including multiple chromogen approaches)
  • Immunofluorescence (including multiple fluorochrome approaches)
  • Simultaneous epitope labeling
  • TUNEL technology (for detection of apoptosis)
  • TUNEL staining and apoptotic index determination
    • Beginning in 2011, the Murphy Lab will no longer quantitate apoptosis.
  • Tissue microarray construction

 

Gene Screening, Detection, and Quantitation

  • In situ hybridization
  • Real time RT-PCR analysis (single gene and gene family chips)
  • Gene family screening
  • Laser capture microdissection (including immuno-guided approaches)

Image Analysis

  • Cell counting
  • Percentages of tissue that stained
  • Use of ImageJ software
Evaluation of Techniques

The purposes for providing the following descriptions and evaluations of the techniques used in Dr. Murphy’s Lab are:

  • to efficiently and fully use our techniques to reach our research goals
  • to improve or modify traditional techniques for new applications
  • to estimate experimental progress for our projects and collaboration projects
  • to select research projects for people who temporarily work in our lab
  • to use the information as a reference

 

Assay How it works Application Limitation(s) How time consuming Labor Cost
Immunohistochemistry (IHC) chromograph-conjugated antibodies bind target proteins in cells and tissue to locate target protein for any cells or tissue which can be attached on slide – not functional study and quantification
– not for living cells and tissue
– not good for secreted protein
+ (days) + +
Immunofluorescence (IF) multiple labeling fluorescence-labeled antibodies bind target proteins in cells and tissue to locate target protein for any cells or tissue which can be attached on slide – not functional study and quantification
– triple labeling is maximum
– not for live cells or tissue
++ (days) ++ +
In situ hybridization (ISH) detect mRNA in cells and tissue to locate target mRNA for any cells or tissue which can be attached on slide – not for functional study and quantification
– cannot do multiple labels
– not for live cells or tissue
+++ (week) ++ +++
TUNEL detect endonuclease-cleaved DNA in cell to identify apoptotic cells in tissue section + (hours) ++ +++
RNA extraction &reverse transcription extract RNA from cells or tissue and reverse transcript RNA to cDNA to prepare cDNA for RT-PCR analysis + (hours) ++ +
Real Time RT-PCR detect gene expression in cells and tissue to quantify gene expression for any tissue and cells – not for location of gene expression ++ (days) ++ ++
Western blot analysis detect protein in cells and tissue to quantify protein expression for any tissue and cells – not for location of protein expression ++ (days) ++ ++
Laser capture microdissection (LCM) laser cuts out small numbers of highly immuno-stained cells from tissue sections to collect pure cell subpopulations for mRNA extraction – cell must be identified under microscope
– protein marker must be specific for captured cells
+++ (days) +++ +++
Soft agar assay culture cells in soft agar (3D culture) for analysis of clonogenic growth – in vitro study +++ (weeks) + ++
Skin invasion asay seed tumor in skin within culture and let them grow for tumor invasion assay – grow no longer than 3 weeks +++ (weeks) + +
Mouse xenograft experiment tumor grows in mouse to study tumor cell behavior in mouse – not for early stage of tumor grow +++ (month) ++ +++
Cell culture grow cells in culture dish (2D culture) to supply the cells in different culture conditions for further study, such as IHC, IF, RT-PCR, Western blot, soft agar assay, tumor animal injection, etc. – cells are a mix of different phenotypes
– good for cell lines culture
– may not get plentifuly or stable enough primary cells for further study
++ (days) + + to ++
Gene knockdown knockdown target gene in target cell line for target gene functional study – only for cell line +++ (month) ++ +++
Flow cytometry fluorescence-labeled Abs bind target protein on surface of live cells and the labeled cells are sorted and analyzed with flow cytometry machine to sort and analyze cell phenotype – only for cells in solution + (hours) ++ +++