The labeling mix as well as all antibodies are purchased from Boehringer. All conditions and solutions should be totally RNAse free. Use gloves and aerosol barrier tips. Linearize the plasmid and check the digest. Extract twice with chloroform:isoamyl alcohol Let dry with caps open for 10 minutes.
Resuspend in suitable volume of nuclease free water. The RNA should appear as a single band with little degradation product and about 10 times more intense than the DNA band. Remove unincorporated free nucleotides with Quick-Spin Columns. Remove the eluate and centrifuge 5 min again. Put the column in new tubes, add the transcription reaction onto them and spin 15 min.
Run a gel loading 1. Don't let the embryos dry at any stage as the amount of background will increase. It is prefered to leave the embryos in a small volume of the solution and to add the next solution to it. Treat all solutions with DEPC add 0. Filter all solutions to remove particles that will stick to the embryos.
Make all the fixations, rinses, washes until the pre-hybridization step on ice except the proteinaseK treatment. Use gloves and aerosol barrier tips for changing the solutions from the fixation step to the end of hybridization. In Situ hybridization.
Day 1. About 5 ml will be needed for each sample after proteinaseK treatment. Prepare hybridization solution. For 50 ml of hybridization solution dissolve;. Hybridization mix recipe 50 ml :. Once dissolved add:. Filter the solution. Incubate each step for 5 min. Wash 3 times for 5 min. Change to 1ml of 4.
Incubate for 3 min for 6dpc at RT, 5 min for 7. Staining for highly expressed gene requires less digestion, but for low expression genes longer digestion may help to get stronger staining. Make sure to thaw the proteinase K stock completely and vortex to dissolve precipitate at the bottom of the tube. Incubation times have to be optimized for each stock. Wash 2 times with PBSw for 5 min. Wash 3 times with PBSw for 5 min. DAY 2. Repeat the addition of the 2XSSC wash twice more.
Wash twice 10 min each in PBS at room temperature. Wash 5 min in PBSw at room temperature. BMblock-mouse antibody buffer 2. During the blocking step, preabsorbe the antibodies. Dilute the antibody in 1. Use this solution to replace the blocking solution. DAY 3. Fast wash embryos with 0. Do another 5 washes with 5 mls 0.
Wash twice, 30 min. Take out staining solutions to warm in RT. Wash the embryos in AP1 buffer mM Tris 9. Replace with 1 ml BM purple and rock slowly in the dark.
In situ Hybridization (ISH) and Fluorescence in Situ hybridization (FISH) - Creative Diagnostics
Stop staining reaction by washing in at least three changes of PBS. Take pictures after placing back in methanol. BM purple becomes more blue and intense in methanol. These protocols describe non-radioactive methods for in situ hybridization on frozen sections, whole mount embryos and on cultured cells. The protocol for in situs on cultured cells is still being optimised, but the current version is the best we have working so far.
Any queries, comments or suggestions should be directed to Andy Groves at grovesa starbase1. Good Luck!
This depends on both the probe and embedding material used. Details of how to prepare RNAse - free slides are in the Appendix. Warm slides to room temperature and dry at 50deg. C for 15 minutes. Rinse once in DEPC-water. Place slides in an RNAse-free glass trough with a stir bar.
In Situ Hybridization Protocols for the Brain, Volume 47
Add ml 0. Add 0. Turn off stirrer when the acetic anhydride is dispersed and leave for a further 10 minutes. Prehybridise for hours at 60deg. We have found that the most effective way to carry out the hybridisation is in slide mailers. It is a good idea to thoroughly seal the lids of the slide mailers with parafilm to prevent evaporation of probe.
After hybridization, it is not necessary to use RNAse-free buffers. Place slides in a trough with a stir bar. Wash in 1xSSC at 60deg. C for 10 minutes. Wash in 1. Cool slides to 37deg. Wash twice in 2xSSC at 37deg. C for twenty minutes each. Treat with 0. C for 30 minutes. These methods enable mRNAs to be detected with great resolution on a single cell level.
- Guillain-Barré Syndrome.
- In situ Hybridization (ISH) in Preparasitic and Parasitic .
- Detection of mRNA by Whole Mount in situ Hybridization and .
- A: Pre-Treatment of Sections;
- ISH: in situ hybridization protocol?
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