DEVELOPMENT OF A NOVEL METHOD OF HUMAN WHOLE BRAIN AUTORADIOGRAPHY
4.2. USING TAPE AS A BACKING MATERIAL.
Since whole body autoradiography is performed using tape to cut large sections, the obvious place to start was to try and cut sections with an adhesive tape which could withstand low temperatures. For this purpose a 5" wide, transparent adhesive tape (Tesa Tape, Leica) was used. Inital experiments were performed on pig brain sections to conserve human tissue while perfecting the cutting technique.
In all the experiments performed on sections adhered to tape, the following method was used, unless otherwise specified: 2 0pm whole brain sections were cut onto tape at -16°C and briefly brought up to room temperature to thaw- mount before the tape was secured to perspex frames (2 0 x 2 0 cm) and stored at -20°C overnight. The following morning the sections were brought up to room temperature for 1 hour. The frames and tape were placed in specially designed perspex wash baths with 3 litres of Tris-HCI buffer (50mM, pH 7.4) containing 120mM NaCI and 5mM KOI for a 15 minute pre-incubation. When the sections were dry, the frames were placed on glass plates so that the tape rested flat on the glass with the section facing upwards. A 2 hour incubation was carried out by covering the section with InM ^H-paroxetine in Tris-HCI buffer in a drop- by-drop manner to ensure the entire section was covered. 4pM citalopram was included to define non-specific binding. Approximately 15ml radioligand in buffer was required for a human whole brain coronal section.
When the sections were incubating, they were covered with a perspex lid to minimise evaporation of the ligand. The incubation medium was then aspirated off and the sections dipped in Tris-HCI buffer for 1-2 seconds to wash off the excess ligand. Washing was carried out in the wash baths containing Tris-HCI buffer for 2 x 60 minutes. The sections were rinsed in ice-cold, distilled water for 3 seconds and dried under a stream of cold air for 30 minutes. When the sections were partially dry they were placed in a freeze dryer for 1 hour to ensure complete dryness and prevent diffusion of the radioligand during
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In order to prevent the tape from sticking to the radiographic film, a fine cover of talc powder was carefully applied around the sections on the tape using a 1/8" paintbrush. If powder gets onto the section it can block the p particles of the radioligand from reaching the film as well as creating a "static" artifact during development. Once the tape was sufficiently powdered, the sections were stuck to a piece of card and apposed to Hyperfilm-^H for 4 weeks. The films were developed as described in Methods, section 2.5.
4.2.1. Pre-Incubation with bovine serum albumin (BSA)
It was relatively easy to produce 20pm sections of good quality by cutting sections onto tape. Preliminary experiments were performed by incubating pig brain sections with ^H-paroxetine to determine the extent to which the ligand bound to the tape and section. As it was apparent that the tritiated ligand bound to the tape to produce substantial background binding, attempts were made to reduce this binding by pre-incubating the tape and sections for 3 - 5 hours with increasing concentrations (1, 3 and 5%) of bovine serum albumin (BSA).
The BSA concentrations were used following a discussion with Mary-Anne Weaver at Brunei University. She found that pre-incubating with BSA substantially reduced the background in her experiments on radioligand binding in trout. However, despite lengthy pre-incubations with BSA in the current investigation, no discernable difference was made to the background binding.
At this point another tape became available which was a 6" wide, opaque adhesive tape from 3M (810). There did not appear to be any difference in the quality of sections gained from cutting onto either type of tape and therefore the effect of ^H-paroxetine background binding was investigated in the presence and absence of pre-incubation with BSA on the new tape. This time, 1.5 x 1.5cm (2.25cm^) pieces of tape were used without any brain tissue on them to reduce the amount of buffer, BSA and radioligand used. There appeared to be no substantial difference in InM ^H-paroxetine binding to either tape. The
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however the average dpm count for a single 2.25cm^ piece of tape was still more than 25 OOOdpm’s (Fig. 4.1).
Tests revealed that after 2 hours of immersion in buffer, the 3M tape began to distort and the adhesive separated from the tape. The Tesa tape maintained its shape and adhesive qualities even after lengthy exposure to liquid. As a result, the use of 3M tape for this type of autoradiography would not be feasible.
In order to determine whether the radioligand was binding to the adhesive on the tape or the backing material, 10OpI ^H-paroxetine was pipetted onto 2.25cm^ pieces of tape on the adhesive side or backing side. Care was taken not to let the incubation medium come into contact with the reverse side of the tape. Fig. 4.2. shows extensive ^H-paroxetine binding on the adhesive side of the tape, with very little binding observed on the backing side. Since the backing side has no adhesive qualities to hold the sections, another method of preventing the radioligand from coming into contact with the adhesive would have to be found.
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