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I would like to know if this antibody works well on floating slices of rat perfused with 4% paraformaldehyde and without glutaraldehyde. Thanks.
Asked on Apr 05 2001
This antibody is known to work with 3-5% glutaraldehyde perfusion. Protocol is as follows - Perfusion : perfuse intracardially through the aorta using a pump with the following solutions : solution A (30ml) : 200-300ml/min solution B (500ml) : 200-300ml/min Solution A : cacodylate 0.1M, sodium metabisulfite 10g/l, pH = 6.2 Solution B : cacodylate 0.1M, sodium metabisulfite 10g/l and glutaraldehyde 3-5% pH = 7.5 Post fixation : 15 to 30 min in solution B, then 4 soft washes in Tris 0.05M with sodium metabisulfite 8.5g/l, pH 7.5 (solution C). Tissue sectioning : Cryostat or vibratome sections can be used. Reduction step : Sections are reduced with the solution C containing sodium borohydride (0.1M) for 10 min. Then, the sections are washed 4 times with solution C without sodium borohydride. Application of anti-Dopamine antibodies : The final dilution is 1/2,000 to 1/5,000 in solution C containing triton X100 0.1%, plus 2% of non-specific serum. A dozen sections can be incubated with 2ml of antibody solution overnight at 4°C. Then the sections are washed 3 times (10 min) with solution C. N.B. : Antibodies may be used at a higher dilution. The customer should explore the antibody dilution to reduce the possibility of high background. Note that a substitution in the buffer system as used in our protocol may change the background and the antibody recognition. PAP procedure : Secondary antibody : Sections are incubated with 1/100 dilution of goat anti-rabbit in solution C for 3 hours at 20°C or 1 hour at 37°C. Then washed 3 times (10 min) with solution C. PAP : Sections are incubated with 1/1,000 dilution of rabbit peroxidase anti-peroxidase complex in solution C for 1 hour at 37°C. Then wash 3 times (10 min) with solution C. Detection : Antibody-antigen complexes are revealed using diaminobenzidine (25mg/100ml) (or other chromogen) dissolved in Tris 0.05M and filtrated; 0.05% of H2O2 is added. The sections are incubated for 10 min at 20°C. Reaction is stopped by transfering sections in 5ml of Tris 0.05M.
Answered on Apr 05 2001