Choice of BufferImmunogold Labelling Protocols - No.8

Antibody incubations - choice of buffer

The buffer must provide the optimal environment for antibody-antigeninteractions while preventing non specific reactions taking place. The buffershould thus contain a balanced salt solution at an optimal physiologicalconcentration and contain additives that appose reactions from hydrophobic,charge, aldehyde and non specific receptor binding.

A typical buffer is as follows:

Phosphate buffered saline (PBS) or Tris buffered saline (TBS) + 1% BSA +0.1% Tween 20 + 1% normal serum (of the second antibody species).Adjusted with HCl / NaOH, preferably to pH8-8.5

(a) PBS or TBS provides the physiological salt solution

(b) 1% BSA Blocks the sections against non specific attraction or proteins (e.g.by aldehyde groups), thus preventing non specific antibody binding

(c) 0.1% Tween 20 prevents hydrophobic attraction of gold or proteins to thesection

(d) 1% Normal serum is from the second antibody species (e.g. normal goatserum if goat anti-mouse gold conjugates are used). This prevents goldlabelled goat antibodies from being bound by receptors for goat antibodies inthe section

(e) The solution is adjusted optimally to pH8 - 8.5. this produces a net negativecharge on the tissue section proteins and prevents non specific binding ofnegative gold particles. If necessary raise the pH even further to pH9 if thereare many positively charged components such as collagen and elastincontaining many lysine groups.

Blocking before antibody incubations

Tissue sections may be blocked before the primary antibody is applied usingthe following solution:

(a) PBS or TBS + 10% normal serum from the second antibody species

or

(b) Neat normal serum from the second antibody species if the section requiresit.

Block for 30 minutes at room temperature.

Blocking prevents the non specific binding of proteins (e.g. by excessaldehyde groups) and by tissue receptors for antibody.