Identification of Specific Relaxin: MATERIALS AND METHODS(4)
Immunohistochemical Localization of Biotinylated Relaxin
Immunohistochemical localization of biotinylated relaxin followed the procedure of Min and Sherwood. In brief, the tissue slides were brought to room temperature, and subsequent immunohistochemical procedures were performed at room temperature. Tissue slides were incubated for 30 min in 50 mM glycine in PBS (pH 7.3) and then incubated for 3 h with blocking buffer 1 (1% BSA fraction V, 0.2% fish gelatin [BBInternational, Cardiff, UK], 5% normal pig serum, and 2 mM NaN3 in PBS). Tissue slides were incubated for 4 h in incubation buffer 1 (1% BSA fraction V, 0.2% fish gelatin, 1% normal pig serum, and 2 mM NaN3 in PBS) in four different ways. flovent inhaler
The first treatment incubated each tissue with biotinylated relaxin probe (4 ^g/ml) in order to localize relaxin-binding sites. The second treatment incubated each tissue with unmodified porcine relaxin (4 ^g/ml). Since the unmodified relaxin does not contain biotin, this treatment was used as a negative control. The third treatment incubated each tissue with biotinylated relaxin plus a 2000-fold excess of recombinant human insulin (Humulin R; Eli Lilly, Indianapolis, IN) in order to determine hormonal specificity of binding of the biotinylated relaxin probe. The fourth treatment incubated each tissue with biotinylated relaxin plus a 2000-fold excess of porcine relaxin in order to determine whether there are finite numbers of relaxin binding sites in the tissue.
Tags: Cells, Female, Human, Relaxin