非放射性杂交方法 Non-Radioactive ISH 英文.pdf

11/20/2002 Non radioactive in situ hybridization The following is a non-radioactive in situ protocol for plants, using an RNA probe. It is derived from several protocols, compiled and worked out by Cindy Lincoln and then slightly modified by myself. The fixation/dehydration/embedding portion is derived from a protocol from Elliot Meyerowitzs lab. The RNA probe synthesis is from David Jackson. The actual in situ section is from both David Jackson and Vivian Irish. An excellent reference for this protocol is: Jackson, D. (1991). In-situ hybridisation in plants. Molecular Plant Pathology: A Practical Approach. Eds. Bowles, D. J., S. J. Gurr and M. McPherson. Oxford University Press. Good Luck, Jeff Long I. Fixation/Dehydration/Embedding DAY 1: Fixative: 4% (w/v) paraformaldehyde; 4% (v/v) DMSO; in 1xPBS Make up required amount of 1xPBS and pH to 11 with NaOH. Heat to 60-70°C. Add paraformaldehyde (in fume hood). Paraformaldehyde should dissolve within a minute or so. Place on ice. When cooled to 4°C, pH to 7 with H SO . Add DMSO to 4% (v/v). 2 4 Collect tissue into ice-cold fixative. Apply vacuum to samples until paraformaldehyde starts to bubble. Hold vacuum for 15 min and release slowly. Repeat until tissue begins to sink. Replace fixative and gently shake overnight (~12 hrs) at 4°C. NOTE: Some tissue will never sink using this method (e.g. flowers) because of air spaces in the tissue. DAY 2: All steps at 4°C and shaking __ 1X PBS wash 30 min __ 1X PBS wash 30 min __ 30% EtOH 60 min __ 40% EtOH 60 min __ 50% EtOH 60 min __ 60% EtOH 60 min __ 70% EtOH 60 min (you can stop here and store tissue for several months in 70% EtOH) __ 85% EtOH 60 min __ 95% EtOH+eosin (until light pink; to visualize tissue) overnight DAY 3: Room temp and shaking __ 100% EtOH+eosin 30 min __ 100% EtOH+