In situ Hybridization of Laminin Chain

In situ hybridization for each laminin chain was performed as described previously [20 (link)]. Briefly, to make complementary RNA (cRNA), DNA fragments were amplified from cDNA of adult rat pituitary using gene-specific primers (for primer information, see [20 (link)]). A BLAST search to verify the specificity of probe sequences confirmed that each probe sequence was specific to the target gene. DNA fragments were ligated into the pGEM-T vector (Promega, Fitchburg, WI, USA) and cloned. Gene-specific antisense or sense digoxigenin (DIG)-labeled cRNA probes were made by using a Roche DIG RNA labeling kit (Roche Diagnostics, Penzberg, Germany). DIG-labeled cRNA probe hybridization was performed at 55°C for 16–18 hr. Each mRNA type was visualized with alkaline phosphatase-conjugated anti-DIG antibody (Roche Diagnostics) by using 4-nitroblue tetrazolium chloride and 5-bromo-4-chloro-3-indolyl phosphate (Roche Diagnostics). Sections treated with DIG-labeled sense RNA probes were used as a negative control to confirm probe specificity. No signal was detected in any sense probes. Sections were observed using an AX-80 microscope (Olympus, Tokyo, Japan).