The slides were incubated overnight with the appropriate probe in moist chambers at 56°C for IGFBP-4 or 53°C for IGF-I. On the following day, the slides were treated with RNA-se-A and rinsed in a series of washes of increasing stringency. The final wash was in 0.1-strength SSC at 69°C or 66°C for IGFBP-4 or IGF-I, respectively. Slides were then dehydrated in alcohols and air-dried. Autoradiography was performed by coating the slides with Kodak NTB2 emulsion (Eastman Kodak, Rochester, NY) and exposing them for 5 days (IGFBP-4) or 2 wk (IGF-I) at 4°C. The slides were then developed in Kodak D-19 developer and counterstained with cresyl violet. buy flovent inhaler
Enzyme Histochemistry for Alkaline Phosphatase Activity
Thawed cryosections (14 |i,m) were rinsed twice in 0.1 M phosphate buffer, pH 7.4. Slides were incubated in Vector Red Kit 1 (Vector Laboratories, Burlingame, CA) substrate reaction solution, according to the manufacturer’s instructions. The slides were incubated for 1 h at room temperature in the dark, rinsed briefly in water, and sealed with a glass coverslip using an aqueous mounting media. To block endogenous alkaline phosphatase activity, levamisole was added to the substrate reaction solution.