Changes

*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.  

*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.  

***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==