Changes

===cDNA ISH===

===cDNA ISH===

Reference:  

Reference: From Rivera et al, 2009, PMID 19168037

[preparation of cDNA probes from restriction fragments or PCR products, using random-priming reaction, e.g. HiPrime?]

Prepare cDNA probes by [[cDNA Random Priming]].

Free-floating tissue sections were collected into 20 ml glass scintillation vials containing ice-cold 2 X SSC (0.3M NaCl, 0.03M sodium citrate) for in situ hybridization. The SSC in each vial was pipetted off, and sections were then suspended in 1 ml of warm prehybridization buffer (50% formamide, 10% dextran sulfate, 2 X SSC, 1X Denhardt’s solution, 50mM dithiothreitol, 0.5 mg/mL denatured salmon sperm DNA) and placed in a 48°C water bath. Two h later, 35S-dCTP-labeled cDNA probes (10 x 10^6 CPM/vial)  were added to the vials and hybridized overnight at 48°C. After overnight hybridization, the sections were washed at 15-min intervals in decreasing concentrations of SSC (2X, 2X, 1X, 0.5X, 0.25X, 0.125X, 0.125X) at 48°C. After washes, the tissue sections were stored in 0.1M phosphate buffer at 4°C and then mounted on gelatin-subbed slides, air-dried, and apposed to Kodak Biomax autoradiographic film (Eastman Kodak Co., NY,

Free-floating tissue sections were collected into 20 ml glass scintillation vials containing ice-cold 2 X SSC (0.3M NaCl, 0.03M sodium citrate) for in situ hybridization. The SSC in each vial was pipetted off, and sections were then suspended in 1 ml of warm prehybridization buffer (50% formamide, 10% dextran sulfate, 2 X SSC, 1X Denhardt’s solution, 50mM dithiothreitol, 0.5 mg/mL denatured salmon sperm DNA) and placed in a 48°C water bath. Two h later, 35S-dCTP-labeled cDNA probes (10 x 10^6 CPM/vial)  were added to the vials and hybridized overnight at 48°C. After overnight hybridization, the sections were washed at 15-min intervals in decreasing concentrations of SSC (2X, 2X, 1X, 0.5X, 0.25X, 0.125X, 0.125X) at 48°C. After washes, the tissue sections were stored in 0.1M phosphate buffer at 4°C and then mounted on gelatin-subbed slides, air-dried, and apposed to Kodak Biomax autoradiographic film (Eastman Kodak Co., NY,