Whole Mount RNA In Situ Hybridization: Zebrafish Embryos
Probe Synthesis
-linearize (10ug) plasmid DNA with the appropriate restriction enzyme (for example: 10 ug
DNA, 4 l 10x buffer, 2.5 l Enzyme, H2O to 40 l, incubate at 37C for 2 hours).
-phenol/chloroform extract (increase total volume to 200 l by adding 160 l of depc H20, then
add equal vol. phenol/chloroform (P/C is stored at 4C as a dual layered solution-take lower
layer under buffer) vortex 20 sec, spin 5 min, transfer upper layer to new tube without
transferring interface)
-chloroform extract (add equal vol. chloroform, vortex, spin 5min, transfer upper layer to new
tube)
-ethanol precipitate (+ 1/10 volume NaAc, then +3 vol 100% EtOH, ice 15 min, spin20 min 4 C,
wash pellet with 100 l 70% EtOH, air dry)
-resuspend in sterile (DEPC) water
-check linearized DNA on an agarose gel
-As an alternative, we perform PCR amplification using the One-Step RT-PCR kit (Invitrogen
and primers that incorporate either T3 or T7 sites; DNA is purified using gel extraction kit
(Qiagen) and used for probe synthesis; This protocol is described in detail by Thisse et al.
-we use ROCHE reagents for probe synthesis
Prepare on ice:
2g linearized DNA
2l 10x transcription buffer
2l 10x DIG or Fluorescein labeling mix
1l appropriate RNAP (SP6, T7, or T3)
1l RNase inhibitor (preferably Rnasin)
DEPC1 water to 20l
Incubate at 37°C for 2 hours
Add another 1l RNAP half way through incubation
Add 1l RNase-free DNase and incubate 5min. at 37°C
Add 2l 0.25M EDTA5 pH 8.0 (RNase free) to stop transcription
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SIGMA Post Reaction Purification Columns:
Spin SIGMA column in a collection tube for 15sec. at 750g
Break base of column and remove lid, spin for 2min. at 750g
Place Column in a new collection tube and add RNA sample, spin for 4min. at 750g
Add 2l 0.25M EDTA pH8.0 (RNase free) to final product in the collection tube
Add 1l RNase inhibitor and 1l RNase Out
Add 25l DEPC water
Store probe at –80°C. Always Keep probe on ICE ***
To confirm presence of probe run 2.2M formaldehyde gel2
Day1: Embryo Fixation and Hybridization
-Fix embryos in 4% Paraformaldehyde/PBS, 5 hours. at room temp. or overnight at 4oC
(PFA should be made fresh weekly)
optional: for long term storage, wash 1 x in 100% MeOH, 10 min., then store at 20oC in 100% MeOH. If embryos are stored in MeOH, before proceeding to next
step, rehydrate embryos (~1ml, 5 min per wash) through 75% methanol/25%
PBST, 50%methanol/50% PBST, and 25%methanol/75% PBST washes. (if just
screening a probe, you can get away with 66% methanol/33% PBST and 33%
methanol/66% PBST washes – leads to shrinking the embryo) work with methanol
and PFA should be done in the hood and waste should be put into its respective
hazardous waste containers.
-Wash 5 x 5 min. at RT in PBST (PBS/0.1% Tween-20) – dechorionate after second PBST
wash if needed (if skipping the MeOH step, embryos can be left in PBST for approx 1 week).
-Permeablize older embryos:
-treat with 10 ug/ml Prot. K in PBST, 5-20 min at room temp, depending on age
eg: 30s for 2-4 somite stage embryos, 1min for 9-13 somite stage embryos, 3 min for 1824 hour embryos, etc.
-refix in 4% paraformaldehyde/ 1 X PBS for 20 min. at room temp.
-rinse 5 x 5 min. in PBST
-Prehyb in 500l hyb mix1 for at least 1 hour at 65oC
-Add 100ng labeled RNA probe to hyb. mix (as a first approx., dilute probe 1/200 in hyb mix.)
-Remove prehyb and add warmed hyb mix plus probe to embryos.
-Hyb overnight at 65oC; for challenging probes (expressed at low levels, perform 40 hour hyb)
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Day2: Washes
Notes:
a) Prewarm washing solutions before adding to embryos.
b) hyb mix containing probe can be removed, stored at –20˚C and re-used many times without
any loss of signal. I have re-used probe >20 times over the course of well over one year.
c) For the following washes, you can use hyb mix that does not contain tRNA or heparin if you
wish to conserve these reagents. Use ~1ml of solutions for the washes
-Wash 5 min in 66% hyb mix, 33% 2 x SSC at 65oC.
-Wash 5 min in 33% hyb mix, 66% 2 x SSC at 65oC
-Wash 5 min in 2 x SSC at 65oC
-*Wash 1 x 20 min in 0.2 x SSC +0.1% Tween-20 at 65oC
-*Wash 2 x 20 min in 0.1 x SSC+0.1% Tween-20 at 65oC (high stringency)
-Wash 5 min in 66% 0.2 x SSC, 33% PBST at room temp.
-Wash 5 min in 33% 0.2 x SSC, 66% PBST at room temp.
-Wash 5 min in PBST at room temp
* Timing is critical
Anti-digoxigenin-Alkaline Phosphatase Binding (first antibody)
-incubate in blocking solution (PBST plus 2% sheep serum, 2 mg/ml BSA [bottle in the fridge,
let it warm up to room temp before opening] – make new block each time.) 1 hour at room temp.
-prepare anti-digoxigenin-AP antibody by diluting it 1/5000 in blocking solution
(anti-fluorscein: 1/10,000)
-incubate in antibody on a shaker for 2 hours at room temp, or overnight at 4oC. (If you leave
overnight , do a couple extra PBST washes)
-wash 5 x 15 min in PBST (may leave in one of the later PBST washes at 4oC overnight on a
shaker, or proceed directly to the next step)
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Day 3: Coloration Reaction (NBT/BCIP) see alternative below
-wash 4 x 5 min in Coloration Buffer2
-mix 45 l nitro-blue tetrazolium (NBT) stock with 10 ml Coloration Buffer, then add 35 l 5bromo-4-chloro-3-indolyl phosphate (BCIP) stock (both available from Roche).
- add 500 l of this mix to embryos and incubate in the dark at room temperature until a blue
reaction product is visible. (can speed up the coloration by incubating at 30oC, or slow by
incubating at 4oC)
-If doing second coloration, rinse 4x with water and proceed to next page. If only doing single
color, stop reaction by quickly washing embryos 2-4 x stop solution (PBST ph5.5), then 2 x 15
min washes in stop solution. Store in stop solution.
BM Purple coloration
We find that BM Purple develops rapidly and has lower background than home-made NBTBCIP. BM purple is (we think) NBT/BCIP dissolved in a proprietary buffer. This buffer is
incompatible with color buffer. As such, the protocol is different.
-After completing PBST washes, perform two quick water rinses
-Resuspend in 1 ml BM Purple (if feeling wealthy, you can use this as a wash step and add a
second 1 ml of BM purple – we usually just incubate in this coloration buffer)
-Allow to color and stop as above using stop solution.
Second Coloration Reaction using INT Red/BCIP
-directly after rinsing first coloration reagent off with sterile water, incubate embryos in 500 ul
0.1M Glycine pH2.2 for 10minutes
-wash 4x 5 min in PBST
-incubate in blocking solution 1 hour at room temp
-incubate in antibody (1:10,000 dilution of alkaline phosphatase-conjugated anti-fluorescein by
Roche) for 2 hours shaking at room temp OR preferably overnight at 4 C (for probes which
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aren’t working well, you can use a 1:5000 dilution of antibody, but this can result in higher
background)
-wash 5x 15 min in PBST
-Wash 4X 5 minutes in Coloration Buffer (100mM Tris pH 9.5, 50mM MgCl, 100mM
NaCl, 0.1% Tween-20)
-Make Coloration Solution:
To 5 mls of Coloration buffer, add 17.5ul INT Red (Iodo-Nitrotetrazolium Violet
Sigma I-8377 at 55mg/ml in DMF. DiMethyl Formamide: Sigma D-8654) and
17.5ul BCIP (50mg/ml).
-To stop reaction: Rinse two times in Coloration buffer. Rinse two times with H20.
Follow with several washes (5) with PBST. Store in 4% PFA at 4C.
Note: INT stock solution should be made fresh every few weeks with fresh DMF.
Change the developing solution a couple of times a day or if it turns pink. If the
solution gets too concentrated it will deposit sticky red precipitate on your
embryos.
Deyolking & Mounting
Embryos can be mounted in glycerol or dehydrated and mounted in permount, with or without
the yolk. Glycerol causes less shrinkage, but is not as effective a clearing agent. If embryos are
mounted on the yolk, they should be kept in the dark, and photographed immediately, because
the yolk darkens rapidly (use double or triple bridged coverslips, so you don’t crush the embryo).
Also, methanol (used for dehydrating the embryos before mounting in permount) makes Fast Red
disappear, so embryos stained with Fast Red can only be mounted in glycerol.
Glycerol mounting (used most often):
-de-yolk the embryos with insect pins while still in PBST
-wash in 30% glycerol/ 70% PBS for about 15min. or until sunk to bottom of tube
-wash in 50% glycerol/ 50% PBS for about 15min. or until sunk to bottom of tube
-wash in 70% glycerol/ 30% PBS
-prepare a slide (25mm x 75mm) with 4 “posts” of high vacuum grease
-transfer the embryos in a glycerol droplet to the coverslip.
-using an insect pin grab the tail and drag the embryo to where you want it on the slide.
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-place a coverslip (22mm x 22mm) on top (careful not to push too hard)
-fill to the edges with 70% glycerol
- Store @ 4C
Mounting in Permount (permanent storage, prettiest pictures, causes embryo shrinkage):
-deyolk
-dehydrate the embryos through an increasing methanol/PBS series: 30%, 50%, 70%,
100% (5min each)
-clear the embryos by transferring into 66% benzyl alcohol/ 33% benzyl benzoate
-once the embryos have sunk in the tube (2-3 mins), pick them up with a glass hair and
place in a drop of permount on a bridged coverslip
-gently lower another coverslip on top and roll until the embryo is in a desired orientation
-embryos mounted in permount can be stored indefinitely at room temperature.
Reagents
1. Hybridization Solution
(store @-20 oC)
50% formamide*
5 x SSC
50 ug/ml heparin
500 ug/ml tRNA**
0.1% Tween-20
sterile H20
0.092M citric acid
(to adjust pH to 6.0)
2. Coloration Buffer
(make fresh each use, in plastic. Must be
clear, not cloudy)
for 50 ml
25 ml
12.5 ml (20XSSC)
50 ul of 50 mg/ml
500 ul of 50 mg/ml
250 ul of 20%
to 50 ml
460 ul of 1M
100 mM Tris-HCl, pH9.5
50 mM MgCl2
100 mM NaCl
0.1% Tween-20
sterile water
For 50 ml
5ml of 1M
2.5 ml of 1M
1 ml of 5M
250 ul of 20%
to 50 ml
*Fomamide is deionized, so once you open the bottle you need to use it all up. Therefore, make
4 50ml conical tubes of Hyb.
**tRNA is really expensive, so only put in one of the four tubes. Don’t use the tRNA tubes for
the washes, only for the prehyb and hyb solns.
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