Optimized concentration of primary antibody incubation, concentration, incubation time, and temperature all affect stain quality.
These variables need to be optimized for each antibody and sample in order to achieve specific staining and low background. Optimization is usually to keep the incubation time and temperature constant while changing the antibody concentration to determine when to get the best signal and low background noise. Long incubation times are often used to ensure complete penetration of the antibody into the tissue sections used in stereological techniques. In order to promote specific staining, longer incubations are usually carried out at low temperatures.
Initial conditions for primary antibody incubation
The choice of primary antibody is quite important when designing IHC/ICC experiments. All steps of the IHC/ICC experiment must be optimized to observe specific staining and to minimize non-specific background staining. This includes conducting a pre-experiment to determine the appropriate incubation conditions for each primary antibody. The dilution of the antigen-affinity purified polyclonal antibody is usually lower than that of the monoclonal antibody, but these values ​​must be determined empirically. In order to obtain reliable and specific signals, high-quality antibodies with minimal cross-reactivity should be used.
These variables need to be optimized for each antibody and sample in order to achieve specific staining and low background. Optimization is usually to keep the incubation time and temperature constant while changing the antibody concentration to determine when to get the best signal and low background noise. Long incubation times are often used to ensure complete penetration of the antibody into the tissue sections used in stereological techniques. In order to promote specific staining, longer incubations are usually carried out at low temperatures.
Initial conditions for primary antibody incubation
The choice of primary antibody is quite important when designing IHC/ICC experiments. All steps of the IHC/ICC experiment must be optimized to observe specific staining and to minimize non-specific background staining. This includes conducting a pre-experiment to determine the appropriate incubation conditions for each primary antibody. The dilution of the antigen-affinity purified polyclonal antibody is usually lower than that of the monoclonal antibody, but these values ​​must be determined empirically. In order to obtain reliable and specific signals, high-quality antibodies with minimal cross-reactivity should be used.
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