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Transfection and Immunofluorescence Staining
LacZ plasmid reporter and the GFP are used as controls to assess the efficiency of the transfection procedure. Within twenty four to forty-eight hours of transfection, expressions of plasmids should be evident. Positive control can be co-transfected with plasmids of interest or maintained in a different well.
To corroborate the specificity of primary Antibody binding, and to cancel out cellular interactions of proteins, a staining control is very relevant. Isotype Antibodies are used to because they retain characteristics that are nonspecific and have no target cells’ specificity.
Alternatively, phalloidin can be used for staining in place of the Antibodies for the staining. In cells, both nonmuscle and muscle, labeled conjugates of phalloidin tend to bind in a ratio that is stoichiometric; a phallotoxin per every unit of actin, because of their similar affinity for minute and large filaments. But unlike the Antibodies’ action against actin, the phalloidin do not attach to the monomeric G-actin.
Advantages of the GFP
The Green Fluorescent Protein (GFP) has assumed a central role as a tool in visualization of both temporal and spartial patterns. There are two major reasons for this. The first is that it helps avoid artifacts because the samples do not need to be fixed to infuse the membrane to permit the Antibodies’ diffusion.
Unstable variants are used in the time dynamics determination of protein expressions, but stable variants are used in the location of specific structures. GFP has variants with stability differences ranging from relatively low to rather very high thus is important in both the cases.
DAPI, as a nucleic acid stain, preferentially stains the dsDNA. With little cytoplasmic labeling, DAPi specifically stains the nuclei. When DAPI staining has been combined with primary and secondary antibodies of choice, signals are amplified, and the choice of isotypes are increased (up to five) to be simultaneously distinguished.
In Kabelitz, D., & In Kaufmann, S. H. E. (2010). Immunology of infection. Amsterdam: Elsevier.