SPI-Mark™ Colloidal Gold Reagents
Instructions for Use and Sample Protocols
Non specific reactive sites on tissues and cell surfaces may need blocking
before antibodies are applied to the specimens. This can reduce potential nonspecific
attraction of the primary or secondary antibodies to the tissue
components or the resin material. Blocking reagents are often used before the
primary antibody step and can be added to the buffer in each step also. They include:
(A) BSA to cover non-specific "sticky" sites of tissue that would otherwise
attract proteins in general. BSA also masks aldehyde groups, which may still
be present in the tissue. Typically BSA blocking uses solutions of up to 10%.
The BSA solution should be freshly made and microfiltered to avoid clumps of
protein attaching to the section and producing unwanted visual contamination.
(B) Normal serum of the second antibody species (e.g. normal goat serum
when using Goat anti-rabbit: gold conjugates). This overcomes the possible
attraction of tissue components for goat antibodies per se. Normal (i.e. nonimmune)
serum should also be microfiltered before use and should be heat
inactivated. It is often used up to 5% in blocking procedures. Unfortunately
normal serums are relatively impure and can add unwanted impurities to the
incubation buffer. They should only be used if necessary. The cross reactivity
between the normal serum and the primary or secondary antibodies may not
always be well defined.
As with all other components in the incubation procedure, blocking reagents
should only be used if necessary. Otherwise they may introduce unknown
signals into the final immunolabeling. It is best to avoid using blocking
procedures until it is found necessary to do so.
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Thursday May 17, 2012
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