4.6 Techniques in Immunopath Flashcards
Techniques used in Immunopathology
Immunofluroescence, Immunohistochemistry, Flowcytometry, PCR Southern DNA alalysis for RLP
Immunoflurorescence
primary ab binds tissue ag, secondary ab directed against the first with a fluorescent marker, wash, light excitationand measure emission of fluorescnece
Immunohistochemistry
primary ab binds tissue ag, secondary ab directed against the first with a enzyme marker (peroxidase, alkaline phosphatase), wash, add substrate to react with enzyme—> this assay usually produces brown pigment in an HE section
advantages of immunohistochemistry over immunofluorescence
immunofluoresence doesn_t last long and also, if you keep light too long it can bleach the tissue. So immunohisto is better bc it is permanent and lasts long.
Flow cytometry
directly labled fluorescent Ab, run the cells in a signel file through a column and excite with light, and measure the emission of light scattered. This is good bc you can reduce bacground noise and detect high ag levels. You can sort these cells
why is immunofluresces/histo more sensitive than flow cytometry
bc multiple secondary ab will bind the primary ab so you’ll see more fluoresence even if there is only a little
the data you get from flow cytometry
the intensity peak of the control represents cells not binding to to what you want, so your sample will have some of the cells appear at this peak, then later at a higher intensity you’ll have the cells of interest that hve the ag of interest, and at even higher intensities you’ll have peaks where there is alot more Ag that was present on those cells for the binding of your ab.
when you comapre cell populations
the dots on your graph do not represent individual cells but realative numbers of cells
Southern analysis
DNA is cut with restriction endonucleases and put on a gel
PCR
primers to a sequnce that you know, heat, add a heat stable polymerase, cool, repeat. Put an excess of primers
