Image | Immunostaining for Dendroaspis natriuretic peptide. Left, Normal human heart. Middle, Human with congestive heart failure (CIIF). Right, Staining with nonimmune response serum (NRS) from same heart as shoen in middle panel.Schirger et al. Mayo Clin Proc. 1999 Feb;74(2):126-30. Protocol for Immunohistochemistry Immunohistochemical studies were performed by the indirect immunoperoxidase method. Tissues were immediately fixed with 10% buffered formalin and embedded in paraffin; sections 6 μm thick were cut and mounted on silanized glass slides. The slides were incubated at 60°C and deparaffinized with graded concentrations of xylene and ethanol. To block the activity of endogenous peroxidase, we incubated the slides with 0.6% hydrogen peroxide in methanol for 20 minutes at room temperature. After being washed, sections were incubated in 5% goat serum (Dako Corp., Carpinteria, California) for 10 minutes at room temperature to reduce non-specific background staining, and they were then incubated with polyclonal rabbit anti-DNP (Phoenix Pharmaceuticals) antiserum at a dilution of 1:500 (in normal goat serum) in humidified chambers for 24 hours at room temperature. All slides were incubated for 30 minutes with a second antibody-horseradish peroxidase conjugate (BioSource, Camarillo, California). The reaction was visualized by incubating the sections with freshly prepared reagent that contained 3′-amino-9′-ethylcarbazole (Sigma Chemical Company, St. Louis, Missouri) in dimethylformamide and sodium acetate. The sections were counter-stained with hematoxylin, coverslipped, and reviewed with use of an Olympus microscope. Six independent observers, without knowledge of the respective groups from which these tissues originated, reviewed these sections. The presence of DNP-LI was quantified on the basis of the following scale of staining: 0=none; 1=minimal density; 2=mild density; 3=moderate density; and 4=maximal density. Control sections were stained with 1% non-immune goat serum Schirger et al. Mayo Clin Proc. 1999 Feb;74(2):126-30.
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