Immunohistochemistry

The following protocols are based upon methods published by Polak & Van Noorden (1997) and manufacturers’ protocols for use with the Vectastain Elite™ ABC kit (Vector Laboratories).

2.51 Antigen retrieval

Formalin-fixed, wax-embedded tissue was chosen for immunohistochemistry, primarily for the superior preservation of tissue morphology achieved using this fixation method compared with fresh-frozen tissue and, secondly, because of the ready availability of samples of control and sclerotic hippocampi, routinely fixed in this manner, in hospital neuropathology departments. This method of fixation would not, however, be

considered optimal for preservation o f the antigenicity of target protein sequences and antigen retrieval by microwaving was investigated as a means o f improving signal. Heating rather than the microwaving process is thought to provide energy to disrupt hydroxyl bonds formed between the protein antigens and the fixative and the citrate salt solution acts to chelate tissue-bound calcium ions, also released by the heating process, which contribute to tighter bonds with the fixative. Fixative bonds are thus permanently broken theoretically revealing protein antigens for interaction with the primary antibody.

Paraffin wax-embedded sections were dewaxed in xylene ( 2 x 5 min) and rehydrated through a series of decreasing ethanol concentrations to distilled water. Slides were labelled appropriately and placed in racks immersed in citrate buffer 0.0IM, pH 6.0 (pH with NaOH) in 300ml plastic containers. The containers were sealed with perforated plastic lids and microwaved individually at full power (750W) for various times, ranging from 0 minutes to 15 minutes, in 5 minute bursts. After each five minute burst, the level of buffer was checked and, if necessary, topped up with fresh buffer. Immediately following microwave treatment, the containers were transferred to a sink and cold tap water was run into the container for 5 minutes until the solution was cool enough to allow removal of the racks without danger of rapid evaporation, drying of sections and precipitation of buffer salts. Optimal antigen retrieval times by microwave were determined for each primary antibody over a range o f concentrations in preliminary studies using replicate tissue sections from a control hippocampus block from one individual.

2.52 Tissue pre-treatment and application o f antibodies

Following antigen retrieval, sections were transferred to PBS (0.0IM phosphate- buffered 0.9% NaCl, pH 7.0-7.4). The pH of the buffer was critical as there is a risk of antibodies becoming detached from the sections at pH<7.0. Due to the nature o f the detection system chosen, it was necessary to block any endogenous peroxidase activity in the sections and this was achieved by incubation with an excess of the enzyme’s substrate, hydrogen peroxide (0.3% in PBS) for 30 minutes. This was followed by a rinse in fresh PBS. Sections were then incubated horizontally with normal (non- immune) serum from the animal in which the secondary antibody has been raised (1:20 in PBS for 30 minutes), in order to saturate any non-specific binding sites in the tissue.

Normal serum contains enough natural antibodies and other proteins to occupy Fc receptors on the two identical heavy polypeptide chains comprising the constant fragment (Fc) of the species IgG, and hydrophobic and electrostatic binding sites, preventing attachment of the specific antibody. These binding reactions are weak, so the serum was not washed off the tissue but merely drained off.

It was important when using an indirect method of immunohistochemistry, with a secondary antibody, that normal serum from the species used to raise the secondary antibody was used to block non-specific binding. If serum from the species producing the primary antibody was used, it would saturate non-specific binding sites but would provide extra deposits of immunoglobulin antigen recognised by the secondary antibody and result in high background staining.

The slides were then placed in petri dishes with a piece o f damp tissue paper to maintain humidity and the sections were outlined with a wax pen. Primary antibody was applied (lOOp.1 per section) and the lids placed on the petri dishes. The sections were allowed to incubate with primary antibody overnight at 4°C. An optimal dilution o f primary antibody was selected on the basis of preliminary studies using replicate tissue sections from a control hippocampus block from one individual. A range of concentrations were used from 1:1000 to 1:10,000 primary antibody in PBS/normal serum (1:20), with the optimum dilution being the highest at which available antigen is saturated leaving some free antibody in solution but with minimal non-specific binding. This was determined by visual inspection of stained slides by microscope following immunohistochemistry.

Two primary antibodies to the GABAa receptor were used in this study, available commercially from Boehringer Mannheim: bd-24 anti-human GABAa a l subunit and;

bd-17 anti-human GABAa (32/p3 subunit. Both antibodies are monoclonal antibodies raised in mice.

Following incubation with primary antibody, the sections were washed with fresh PBS and incubated for one hour at room temperature with lOOpl o f biotinylated secondary, anti-mouse, antibody diluted 1:200 in PBS/normal serum.