All experimental procedures were performed in accordance with the NIH Guide for the Care and Use of Laboratory Animals and a protocol approved by the Children’s Hospital Research Foundation Animal Care Committee.
A rat IGFBP-4 cDNA (444-basepair [bp] Sma I-//mdIII fragment of the coding region obtained from S. Shimasaki, The Whittier Institute, La Jolla, CA), with 94% nucleotide sequence homology to mouse IGFBP-4, was subcloned into the transcription vector pGEM-4Z (Promega Biotec, Madison, WI). Similarly, a 317-bp £coRI-£coRI fragment of a rat IGF-I cDNA, with 94% nucleotide sequence homology to mouse IGF-I, was subcloned into the transcription vector pGEM-7Zf(+) (Promega Biotec). The transcription reaction for both probes was performed with [a-33P]uridine triphosphate (UTP; New England Nuclear, Boston, MA), giving a final probe specific activity of approximately 3.0 X 108 disintegrations per minute (dpm)/|xg and 7.5 X 108 dpm/|xg for IGFBP-4 and IGF-I, respectively. asthma inhalers
In Situ Hybridization
The in situ hybridization procedure was performed as previously described. Briefly, tissues were fixed in 4% paraformaldehyde, washed in PBS, and then infiltrated with 30% sucrose. The tissues were embedded in O.C.T. embedding compound and stored frozen at -70°C until sectioned by cryostat. Sections (14 jjun) were then post-fixed with 4% paraformaldehyde, treated with 0.25% acetic anhydride in 0.1 M triethanolamine, and rinsed in double-strength saline sodium citrate (SSC; single-strength SSC is 0.15 M sodium chloride, 0.015 M sodium citrate, pH 7.4).