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 veral protocols, compiled and worked out by Cindy Lincoln and then slightly modified by mylf. The fixation/dehydration/embedding portion is derived from a protocol from Elliot Meyerowitz's lab. The RNA probe synthesis is from David Jackson. The actual in situ ction 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
H2SO4. Add DMSO to 4% (v/v).
Collect tissue into ice-cold fixative. Apply vacuum to samples until paraformaldehyde starts to bubble. Hold vacuum for 15 min and relea 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) becau 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人声鼎沸是什么意思
__ 40% EtOH 60 min
__ 50% EtOH 60 min
__ 60% EtOH 60 min
__ 70% EtOH 60 min (you can stop here and store tissue for veral 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
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__ 100% EtOH+eosin 30 min
__ 100% EtOH+eosin 60 min
__ 100% EtOH+eosin 60 min
__ 25% histoclear, 75% EtOH 30 min
__ 50% histoclear, 50% EtOH 30 min
__ 75% histoclear, 25% EtOH 30 min
__ 100% histoclear 60 min
__ 100% histoclear 60 min
__ 100% histoclear + 1/4 volume paraplast chips overnight (no shaking)
DAY 4:
__ place at 42°C until chips melt completely
__ Add 1/4 volume of chips until completely melted
__ Move to 60°C for veral hours
__ Replace wax/histoclear with freshly melted wax overnight (60°C)
DAY 5:
__ __ 2 wax changes (parated by veral hours)
DAY 6:
__ __ 2 wax changes (parated by veral hours)
DAY 7:
__ __ 2 wax changes (parated by veral hours)
DAY 8:
Place tissue in molds. Store at 4°C.
II.Sectioning
Sections are 8µm thick. Slides ud are ProbeOn Plus from Fisher Biotechnology. They are pre-cleaned and charged. They also have a white frosting on them that allows you to sandwich them together later in the hybridization/detection steps of the protocol.
Pre-warm slide warmer to 42°C. Place slide on RNa-free slide warmer and apply veral drops of DEPC-treated water. Float ribbon of tissue ("shiny" side down) on top of water and allow to sit for a few minutes (we u paintbrushes to handle the ribbons). During this time the ribbon should flatten out.
Drain off water using a Kimwipe (excess water will cau bubbles). Allow slides to incubate on slide warmer overnight so that tissue adheres to slide. Sectioned tissue can be stored with desiccant for veral weeks at 4°C.
III.In vitro transcription
Linearize plasmid to give run off transcripts with the appropriate polymera. U an excess of enzyme/time (i.e. 5X/4hours) to ensure cutting is to completion. Do not u enzymes that leave a 3' overhang. Phenol/chloroform extract twice and precipitate to get rid of any RNas.
O. Set up transcription reaction as follows: Resuspend at 0.5µg/µl in DEPC-treated H
2
DNA 2 µg 4µl
5x buffer 5µl
5Xnucleotides 5µl
RNa inhibitor to 1unit/µl
RNApolymera to 0.4 units/µl
H20 to total
TOTAL 25µl
Incubate at 37°C 30 to 60 minutes.
Run 1 µl on a minigel at <100V for roughly 15 minutes as RNA degrades quickly (treat gel box and comb with 0.2N NaOH for 30 minutes before u). Add 75 µl H
0, 1 µl 100mg/ml tRNA
2
Ac and 2 5units RNa-free DNa. Incubate for 10 minutes at 37°C. Add equal volume 4M NH
4 volumes EtOH. Precipitate at -20°C.
Spin down RNA pellet (it should be large). Rin pellet with 70% EtOH. Air dry or speed vac with no heat.
5X nucleotides are 2.5mM each in H
O. We u a 1:1 dig-UTP:regular UTP. We get our
2
unlabelled nucleotides from Stratagene, and our dig-UTP from Roche.
Carbonate hydrolysis:
You now want to "chop" up your probe into pieces between 75 and 150 bp long. We typically calculate the reaction for 150 bp final length.
The formula is as follows:
Time=Li-Lf/(K•Li•Lf)
日月如Li=initial length of probe (in kb)
Lf=final length of probe (0.15 kb)
K=0.11 kb-1min-1
O. Add 100 µl 2XCO3- buffer. Incubate at 60°C for calculated time Resuspend pellet in 100µl H
2
Neutralize with 10µl 10% acetic acid. Add 1/10 volume 3MNaAc (pH 5.2) and 2 volumes EtOH. Precipitate at -20°C. Rin pellet with 70% EtOH. Resuspend in 50% formamide at 1µl/slide for convenience.
A crude way to estimate how much probe you have made is to compare the intensities of the RNA band to that of the DNA template band on your gel. If they are equal, you have synthesized roughly 2 µg of RNA probe.
U probe at a final concentration in the hybe of 0.5ng/µl/kb. For a new probe try up to 5X higher or lower to find the best concentration. Fisher Probe On Plus slides require 100µl probe per slide. So, if probe is 0.5kb, you need 0.5X100X0.5=25ng probe/slide. Thus 2µg of probe is sufficient for 80 slides.
IV.In situ ction pretreatment
新娘盘发Make all solutions RNa-free (I don't DEPC-treat most of my solutions or water supply, but I do autoclave everything before I u it.) Treat plastic containers with 0.1M NaOH overnight and rin with sterile water. Bake glassware and stirbars.
1. Deparaffinize/dehydrate ctions:
2X 10 min histoclear
2X 1-2 min 100% EtOH
1-2 min 95% EtOH
1-2 min 90% EtOH
1-2 min 80% EtOH
1-2 min 60% EtOH
1-2 min 30% EtOH爱情的图片
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1-2 min H
2
2. 15-20 min 2X SSC room temp
3. 30 min proteina K (1µg/ml) in 100 mM Tris pH 8, 50 mM EDTA at 37°C
(Prewarm Tris/EDTA solution to 37°C-add proteina K from 10 mg/ml frozen stock immediately before adding slides.)
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4. 2 min 2 mg/ml glycine in PBS room temp
5. 2 min PBS room temp
6. 2 min PBS room temp
7. 10 min 4% (w/v) paraformaldehyde in PBS pH 7 (made fresh) room temp
8. 5 min PBS room temp
9. 5 min PBS room temp
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10. 10 min 0.1 M triethanolamine (made fresh to pH 8) and acetic anhydride.
Elevate slide rack in container of triethanolamine with stir bar. Dispen acetic anhydride (4 mls in 800 mls triethanolamine) into triethanolamine a few minutes before putting slides in so that it is mixed well.
jk罗琳简介11. 5 min PBS room temp
12. 5 min PBS room temp
13. dehydrate:
30 c 30% EtOH
