CHAPTER2
2.6 IMMUNOCYTOCHEMISTRY .1 Neutrophils
2.6.2 Protocol for immunolabelling : synovial tissue
Three-micron sections of the wax-embedded tissue were adhered onto adhesive coated (poly
L-lysine; Sigma, St. Louis) slides. Detection of immunoreactive TK, BI and B2 receptors was performed by standard histochemistry techniques using the relevant specific primary antibody (see section 2.6.1.), and the appropriate anti-species secondary antibody conjugated with peroxidase-antiperoxidase (PAP) immuno-enzyme complex. The final reaction involved
the hydrolysis of diaminobenzidine (DAB), the chromogen substrate, and the product precipitate ( dark brown) was visualised by light microscopy.
The PAP method as modified by Figueroa et al. (1988) was used for the irnmunolabelling of TK and the kinin receptors. The three-micron thin sections of the wax-embedded tissue sections on glass slides were heated on a heating block (Clifton, UK) at 60°C (to melt the wax) for 5 min. The peroxidase-anti-peroxidase (PAP) method required dewaxing the tissue sections in analytical grade xylene (analytical grade; Saarchem, SA) twice for 10 min each, rehydration with graded ethanolic solutions (100 %, 90 %, 70 % and 50 % in distilled water, v/v) and distilled water as the final rehydrant. When the tissue was approximately 50 % rehydrated, it was immersed in absolute methanol (analytical grade, Saarchem, SA) for 20 min to quench endogenous tissue-peroxidase activity. Following rehydration, the tissue was boiled in a metal-salt solution, 0.lM sodium citrate, pH 6.0 (analytical grade, Saarchem, SA) at 80°C for 10 min (antigen retrieval) (Shi et al. 1999) in a conventional microwave oven (R-4A52; Sharp Corp., Japan). The tissue in the boiling solution was allowed to cool down to RT (approximately 20 - 25 min). The tissue-sections were then further blocked with 10 % H202 (analytical grade, Saarchem, SA)/90 % absolute methanol (v/v) for 20 min to quench endogenous peroxidases. Incubation with the primary antibody was performed at 4°C for 18 h in a humidified chamber. Then, the tissue was treated with a peroxidase-anti-peroxidase (PAP) streptavidin-biotin conjugating system (LSAB K0690;
Dako, USA) for 20 min. each. This entailed incubating the tissue with a universal (goat, rabbit, mouse) IgG-biotin link for 20 min at RT in a humidified chamber. Next, the tissue sections were covered with a strepavidin-peroxidase conjugate for 20 min at RT in a humidified chamber. The labelled antibody bound to the PAP immuno-enzyrne complex was visualised by incubating the sections, in the dark, for 5 to 7 min with liquid
diarninobenzidine (DAB) precipitant (DAB K3465; Dako, USA). The sections were counterstained with Mayers Haematoxylin (Sigma, St. Louis,) for 3 min and intensified under running tap water for 5 min. Sections were then dehydrated by a reversal of the dehydration process from distilled water, through the increasingly concentrated ethanolic solutions into xylene, and finally mounted with a permanent medium (Entellen, Merck) (Appendix 2. 7).
Results were viewed by conventional light microscopy under a Nikon photo-microscope (Nikon Optiphot; Nikon, Japan). All incubations were carried out in a humidifying chamber.
Between incubations the sections were washed thoroughly by submerging the slides in PBS [0.01 M phosphate buffer (pH 7.2) containing 0,0027 M potassium chloride, 0.137 M sodium chloride; Sigma, St. Louis] for 5 min. Tissue sections were not allowed to dry out at any time during the labelling process. The labelled slides were stored in the dark.
Positive tissue and method controls/or immunocytochemistry (ICC)
As the kinin moiety within the kininogen molecule has been previously localised on control circulating neutrophils, these cells were used for the positive and method controls.
For the TK studies control human salivary gland tissue was used a positive control tissue to demonstrate localisation, upon previous evidence of abundant labelling for TK in such tissue (Schachter et al. 1980). Samples of fresh control human salivary gland were collected at post-mortem, fixed in 5 % formalin and embedded in paraffin wax. During each immunolabelling run, this tissue, demonstrating the presence of TK in the ducts of the human salivary gland, served as an appropriate method control for the labelling procedure.
Sections of fresh control human kidney collected at post-mortem, served as positive controls for B2 receptors. During each labelling run, this appropriate positive control tissue demonstrated the presence of B2 receptors in tubules and connecting ducts (Naidoo et al. 1996a). Human spinal cord was used as positive control tissue for Bl receptor immunolocalisation. During each labelling run, the presence of immunoreactive BI receptors was demonstrated in the neurons in the substantia gelatinosa of the spinal cord (Raidoo and Bhoola, 1997). All labelled slides were stored in the dark to minimise bleaching of fluorescence and fading of precipitant immunolabels.
Negative method controls for ICC
The absence of positive specific immuno-labelling following pre-adsorption of the primary antibody with an excess of the specific antigen demonstrated the specificity of the antibody utilised. The primary antibody was diluted I :500 with 0.01 M phosphate buffer (pH 7.2) and added to a 2 mg/ml stock solution of antigen to yield a final concentration of 1 mg/ml antigen. This antibody-antigen conjugate was incubated overnight at 4°C to allow maxnnum formation of antigen-antibody complexes. Following removal of the preadsorbed antigen by centrifugation (2200 x g, 4°C, Heraeus Biofuge 1.3 R, Germany) the supernatant was used for the immunolabelling experiments instead of the primary antibody.
For the immunolabelling of controls for the synovial tissue the primary antibody was replaced with goat non-immune serum diluted in 0.0 I M phosphate buffer (pH 7.2)/1 % BSA. In the method controls the primary antibody was replaced with buffer. All dilutions made up in 0.01 M PBS/I% BSA (pH 7.2).
2.7 MEASUREMENT OF TISSUE KALLIKREIN IN SYNOVIAL FLUID