Análisis de la oferta y de las importaciones

Tissue handling.

Tissues were removed quickly and immediately put in ice cold Hanks balanced salts solution, pH 7.5 (GIBCO BRL, Scotland). Unfixed tissues were embedded in Tissue- Tek (Sakura Finetek, Netherlands) and frozen in isopropanol precooled in liquid nitrogen. Cryostat-sections were cut at a thickness described in each experimental chapter and were placed on poly-L-lysine or gelatine coated slides. Tissues for immunohistochemistry were postfixed for 2 min at room temperature in 4% formaldehyde (BDH Laboratory Supply, UK) and 0.03% picric acid in phosphate- buffered saline (PBS), or without picric acid for immunofluorescence studies. Inactivation o f endogenous peroxidase was carried out in 50% methanol and 0.03% H2O2

achieved by pre-incubation with normal horse serum (NHS; Harlan Sera-Lab, UK) at room temperature for 20 min., as described in detail by Llewellyn Smith et al. (1993).

Immunostaining,

An indirect immunohistochemical and immunofluorescent method with three layers o f antibodies was used. P2X receptor antibodies from rabbit were allowed to react with biotinylated donkey anti-rabbit antibody and detected with avidin-coupled horseradish- peroxidase/nickel intensified diaminobenzidine (DAB) or avidin-coupled Texas Red. The P2X antibodies were obtained from Roche Bioscience (Palo Alto, Calif., USA). The P2X subtype-selective antibodies were raised in rabbits against a specific 15 amino acid residue at the carboxy-terminus o f each P2X receptor molecule (Oglesby et al. 1999; antigenic sequences are shown in Table 1).

M ouse antibodies were detected with a second layer o f goat anti-mouse immunoglobulin G (IgG; Sigma, MA, USA) and immunoreactivities were shown with a fluorescein-isothiocyanate (FITC) labelled chicken anti-goat antibody (ICN, Biomed, Calif., USA).

All primary antibodies were diluted in 10% NHS in PBS containing 0.05% thimerosal (TPBS; Sigma), secondary antibodies in 1% NHS in TPBS, and tertiary antibodies in TPBS only. Primary antibodies were incubated on tissue section over night, secondary and tertiary antibodies were incubated for 1 h. Streptavidin conjugates were allowed to react with biotinylated secondary antibodies for 30 min. After each incubation w ith antibodies or w ith streptavidin conjugates the slides were washed 3 times for 5 m in in PBS with gentle agitation.

o f P2X-antibodies was further determined by preabsorption with their corresponding antigenes. Preabsorptions were performed as follows: working-dilutions for each P2X receptor antibody (as indicated in the Specific Materials and M ethods o f each experimental chapter) were incubated with a 1 0 fold excess o f their antigeneic peptide for

Ih at RT and were then left at 4 °C over night. These antibody/ antigene mixes were spun down at lOOOOg for Ih at 4 °C and the supernatants were applied to tissue sections instead o f primary antibody solutions. Incubation times and wash-steps were identical to the procedure described above.

During the incubation o f an antibody with its cognate peptide larger immuncomplexes can form , which may cause unspecific binding with secondary or tertiary antibodies. These big immuncomplexes can be precipitated by centrifugation in a microcentrifuge (at approx. lOOOOg) leaving the small, soluble antibody-antigene complexes in the supernatant. Precipitation o f the big immuncomplexes may lower the concentration o f the prim ary antibody, which is applied to the tissue sections after the preabsorption procedure. However, the concentration o f the prim ary was probably not significantly lowered in the experiments o f the present thesis as some preabsorption experiments showed great reduction, but not abolition o f the immunostaining.

To determine possible crosslinking between rabbit and mouse antisera, single and double staining experiments with each antibody were made.

Tyramide amplification.

Antibodies were applied as for immunohistochemistry (but a lower concentration; between 1/7 to 1/10 o f the antibody-concentration for non-enhanced immunostaining) and a tyramide signal amplification (Renaissance, TSA indirect, NEN, USA) was

performed. The tissue sections were incubated over night with primary antibodies and then extensively washed (5x for 5 min in PBS with gentle agitation). Secondary antibody (biotinylated donkey anti-rabbit) was applied as described above. A fter incubation for Ih slides were again washed 5x for 5 min in PBS with gentle agitation. The biotinylated secondary antibody was coupled to streptavidin-HRP (diluted 1:1500 in PBS) at RT for 15 min followed again by extensive washing o f the slides (2x 5min in PBS; Ix 5min in PBS, 0.05% Triton X-100; 2x 5min in PBS). Then the amplification solution from the kit was applied for 6 - 8 min followed again by extensive washing (2x 5min in PBS; Ix 5min in PBS, 0.05% Triton X-100; 2x 5min in PBS). Streptavidin coupled HRP and streptavidin coupled Texas Red were applied at dilutions (in PBS) o f 1:1500 and 1:200, respectively. The horseradish peroxidase/DAB-stain was analysed by light micrsoscopy, Streptavidin-Texas Red labelling was visualised by fluorescence- and confocal- microscopy, as described in the indivual experimental chapters.

Identification o f cells showing immunolabelling fo r P2X receptors.

Cells were identified by their characteristic shape, localisation within the tissue and colocalisation with specific markers. Counterstaining o f P2X antibodies with different histological stains was performed to identify P2X receptor expressing cells and tissues by their morphology. Different procedures for cell identification are described in the individual experimental chapters.