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*An antibody directed against a specific antigen is directly conjugated with the fluorescent dye and applied to the sample.  
 
*An antibody directed against a specific antigen is directly conjugated with the fluorescent dye and applied to the sample.  
 
'''Indirect staining'''
 
'''Indirect staining'''
*Utilizes a double layer technique- a primary, unlabelled antibody is applied to the sample, followed by a secondary antibody, an anti-immunoglobulin that has been conjugated to a fluorochrome.  
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*Utilizes a double layer technique - a primary, unlabelled antibody is applied to the sample, followed by a secondary antibody, an anti-immunoglobulin that has been conjugated to a fluorochrome.  
 
**Indirect staining has several advantages:
 
**Indirect staining has several advantages:
 
***Several secondary antibodies bind to each primary antibody, so the resulting fluorescence is brighter than that of the direct staining.
 
***Several secondary antibodies bind to each primary antibody, so the resulting fluorescence is brighter than that of the direct staining.
 
***One preparation of secondary antibody can be used to test many sera
 
***One preparation of secondary antibody can be used to test many sera
 
***By using a mixture of primary antibodies, it is possible to detect the relative expressions of different antigens in the same cell
 
***By using a mixture of primary antibodies, it is possible to detect the relative expressions of different antigens in the same cell
***Quite often loss of antibody is sustained during the conjugation- in the indirect method the primary antibodies do not need to be conjugated, so this limiting factor is reduced.  
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***Quite often loss of antibody is sustained during the conjugation- in the indirect method the primary antibodies do not need to be conjugated, so this limiting factor is reduced.
    
==Applications==
 
==Applications==
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