C57BL/6 mice (Harlan) are maintained at St. Jude Children's Research Hospital animal care facility in accordance with the NIH Guidelines for the care and use of laboratory animals. Mice are kept on a twelve-hour light cycle with food and water ad libitum.
Embryonic day 11.5 (E11.5), E15.5, postnatal day 7 (P7) and P42 tissues are collected and fixed with 4% paraformaldehyde (PFA). Following overnight fixation tissue is rinsed with 0.1M phosphate buffered saline(PBS), dissected and cryo-protected overnight in 30% sucrose in PBS. For each time-point tissue is embedded in a Cryomold® (Sakura Finetek USA, Inc.) containing TBS Tissue Freezing Media (Triangle Biomedical Sciences) and frozen on dry ice.
For each block of tissue 16 µm cryo-sections are collected onto 51 SuperFrost Plus slides (Fisher Scientific). A total of 101 sections are collected for the E11 block and P42 coronal block and 151 sections for the E15 block, P7 block and P42 sagittal block. The first section of each block is collected on slide 0. The remaining sections are collected on slides 1-50. Each slide contains sections at approximately 800 µm intervals, i.e. slide 1 contains sections 1, 51, etc. For each set of 51 slides generated, slide 0 and every 10 th slide are stained with cresyl violet to be used as reference slides. The remaining 45 slides are each hybridized with a unique probe. Since each section contains multiple pieces of tissue each probe is hybridized to 55 pieces of tissue representing three planes of section and four developmental ages
Detailed Tissue Processing Protocols
BGEM: An In Situ Hybridization Database of Gene Expression in the Embryonic and Adult Mouse Nervous System
Susan Magdaleno, Patricia Jensen, Craig L. Brumwell, Anna Seal, Karen Lehman, Andrew Asbury, Tony Cheung, Tommie Cornelius, Diana M. Batten, Christopher Eden, Shannon M. Norland, Dennis S. Rice, Nilesh Dosooye, Sundeep Shakya, Perdeep Mehta, Tom Curran
PLoS Biology Vol. 4, No. 4, e86 DOI: 10.1371/journal.pbio.0040086
BGEM Home Page
- Special thank-you to Dr. Tom Curran
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