As the result of the RT-PCR, an approximately 160-base pair (bp) DNA fragment was amplified from messenger RNA of 14.5-dpc ICR mouse placentas. This cDNA fragment was cloned into pCRII vector using a ТА cloning kit (Invitrogen), and its nucleotide sequence was determined using a DyeTerminator Cycle Sequencing system and ABI377 Autosequencer (Perkin-Elmer, Irvine, CA). The size of the PCR product was confirmed to be 158 bp, which was the same size as the corresponding site of calbindin-D9k cDNA of the other animals, and the nucleotide sequence was found to be highly homologous to the rat cDNA with a difference of only 8 nucleotides. This sequence was registered in GenBank with the accession number of MMU88544. Moreover, this sequence had a reading frame, and the translated amino acid sequence was the same as that of rat calbindin-D9k except for 1 amino acid. Hence, we concluded that this cDNA fragment represented the mouse homologue of the calbindin-D9k and used it for further study. buy asthma inhaler
In Situ Hybridization
In situ hybridization (ISH) using digoxigenin (DIG)-la-beled probes was performed as described. DIG-labeled cRNA probes for PDGFRa or calbindin-D9k were synthesized using a DIG RNA labeling kit (Boehringer Mannheim, Mannheim, Germany) from the linearized p-CRII plasmids containing mouse PDGFRa or putative mouse calbindin-D9k cDNA fragments, respectively. Sectioned specimens for in situ hybridization were prepared in the same way as those for immunohistochemistry.