In situ hybridization Protocol

For paraffin sections

Deparaffinization

and

rehydration

of

the

specimens

(RT.)
Xylen- 2 changes, 10 min. each.
100% ethanol- 2 changes, 2 min. each.
95% ethanol- 2 min.
80% ethanol- 2 min.
70% ethanol- 2 min.
50% ethanol- 2 min.

Pre

hybridization.

\ r Rinse 5 min. in H20DDW,DEf>C.

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\ Heat denature 30 min. at 700C in 2* S SC. .-::7
~'rl\'\'(.
!...Rinse5 min. in H20DDW D'iP~"- '\~~'\
~-t Proteinase K digestion 1Q...!:!!iJ. 370C . ,- r
- ~s,
These steps denatures RNA. and probably also removes some
?-~~) .t\1'\
to make RNA in sections more accessible to hybridization.
Post-fix specimens 20 min. at RT. in freshly prepared 4%
Rinse 5 min. in 0,2% glycine at RT. ~.
~ \_
'-"
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Rinse 10 min. 'n 0.25% acetic an~ik
~~'-RT'r - ----.----.))
~
inse 5min. in H20DDW,DEPC.
l,i:l' .
AiT"jdry specimens, making sure specimens are absolutely
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of the proteins
PFA fixative.

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dry.

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Solution
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Hybridization
buffer.
50% Formamid
4*SSCDEPC
1*Denhard' s
0,5 mg/ml salmon sperm
0,25 mg/ml yest t-RNA
10% dextran sulfat

Method
Mix the labeled probe with Hybridization
buffer ( estimating 50-100
III per 22* 22 mm coverslip ) heat to 850C for
min and coolon
ice.
Take slides after air-drying and pipette 100 ~ lover
each group of
sections. Gently put parafilm over each group of sections and incubate
~night
at 600 in sealed box containing paper towels saturated with

5>

5x sSC.~

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Washing

after

the

hybridization.

1. Wash the slides in 5* sse until the coverslips were slide off ..
2. Wash in.2*SSC +50% formamide at 650 e for 30 min, 300 ml for 10-20
slides.
3.2*SSC 3 times for 5 min, t 37 e
~.~:~ashin,,"g solution +. 1 ~/ml RNAz~, t 37 C, 30 min
..,.... -.J~C I:JD~ furmamide l~, .,
_n
6.2*SSC t 37 e 1-5--miR
~\ '" ~ t- t~" f-. 7.~SC
t37 C 15 min
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Immunological

detection

of

DIG-labeled

hybrids

Wash in\TBS 3 times for 3 min each.

/ \~\
10 min ill O.JM HeL in TBS (iHaeti\late-the endogeBoas enzyme ~tiv~
5 ffiiB: iff TES.
~Q\
\t~~t"
Blocking in 3% *"goat serum in TBS, at least ~~ min at RT.~; ~\\~'" ~
Replace the blocking solution by:
alkaline phosphatase
conjugated anti-DIG
antibody diluted 1:500 in 1% goat serum ':CBS for 60 min at RT.
Wash 5-10 times in TBS for 30 min.--)?::~~"e:\~ ! (Q
\~~ r\ - ~\l,I<~" i>To perform th~ col9.~~reaction: add'''', 0 ~l NBT, jif.5 ~l X-phosphate
solution
and 2.4 mg le~isole
to 10 ml buffer containing O.IM Tris pH 9.5, 0.05M .;J
MgC12 and O.lM NaCl.
4-~\ DOW1 oS ..
\ Tv-;<" A
~.B.( 1~'O.w f.$-\ 11M : "<J,.f
\00M- ~ N ..
,f ~M
Incubate in RT for few hours or in 40 e for on or few' days, depend on the
background
and signal.

Preparation

of RNA

probe for

in situ

hybridization

A. Restriction
digest and purification
of the plasmid.
For anti-sense probes (which hybridize to the RNA), linearize the plasmid
containing
the cDNA insert by cutting it with a restriction enzyme at the
original 5-end of the cDN A insert (or outside at the poly linker). Use the
_polymerase
going from the original 3-end of the insert towards its 5-end.
Use the same logic for the sense probes (Le., cut at the side of the 3-end of
the insert
and use the RNA polymerase going towards it).
a. Extracting
once with phenol-chloroform
(i.e., add an equal volume of
Tris equilibrated
phenol-chloroform
mixture,
vortex for 30 seconds spin
for 5 min. and take the UPPER aqueous phase for step Itblt).
b. Ethanol
precipitation
[add sodium acetate
(3M pH 5,2) to a final
concentration
of 0,3M to the a aqueous phase. Incubate at -20oe to 30 mID.
Alternatively
you can use liquid nitrogen for 2 min.Spin for 10 min.].
c. Wash the pellet with 75% ethanol.

d. Re suspend in H20 to yield 1 mg/ml.

e. Verify digest by electrophoresis
on an agarose gel
(usually
1%w/v
in 1 *TAE buffer, add Ethidium bromide (5 mg/ml) to a final concentration of
0,5 mg/ml; i.e. a dilution of the stock solution).
Compare cut
vs. uncut
plasmid vs. marker (for a marker I use the 1 Kb ladder of "BRL
For
detection do not load more than 0,5 mg of the DNA and 1 mg of the marker.
ft

).

50 *T AE running
buffer
242 g
Tris base
57,1 ml
Acetic Acid Glacial
37,2 g
Na2EDTA*2H20
H20 to 1 liter.
5 * gel loading
buffer
50% v/v Glycerol
0,2M EDTA pH 8,0
0,05% w/v Bromphenol Blue

B. The

Labeling

Add the following items in the order they appear: (note,

should be RIBOnucleotides
and NOT the deoxy form)
1. DNA( approx. 0,5 Jlg to 1 Jlg)
2. 5* transcription buffer ( arrives with the enzyme)
3. Dig RNA Labeling mix, 10*conc.
4. DTT (100 rru\t1 )
5. RNase inhibitor( like RNasin )
. 6, RNA polymerase(TI/TI/SP6
)
7. Water
Incubate 2h 37 C.
Next, add to the reaction the following:
1. RNase inhibitor
2. DNase
Incubate 10 min. at 370C
Following this incubation add:
I.EDTA 0,5M ph 8.0
1,25 Jl1
2.LiC14M
3,1111
3.Ethanol( -20 C}
90111
4. Mix well and store at -20 C for 2h or over night.
5. Centriguge at 12000g at 4 C for 15 min.
6. Wash the pellet with 70% ethanol

III

2,5 III

2,5 ).Ll
2,5 III
1 III
1 III
14,5 III

Reagents

and

solutions

4 % paraformaldehyde
in
To prepare 100 ml of fixative:
Add 4 g of paraformaldehyde
Add 6-10 ~l of 10 M NaOH.
Add 10 ml of 10*PBS.
Adjust the volume to 100 ml
Stir at 650C untill the solution
Store at 40C for up to 24 h.

PES.
to 50 ml water.

with water.
will become

clear.

H20DEPC

Add 500 ~l of Diethyl Pyrocarbonate

(DEPC, SIGMA, D- 5758) to 500 ml
H20DDW'
Incubate flask with H20DDW.DEPCin water bath at 370C for overnight.
Sterilize by autoclaving.
Store at room temperature
.~.
All the solutions which used for "IN SITU" prepared with H20DDW,DEPC.

lO*TBS

t!)A

~J~~

Dissolve 1S'M g of NaCl and 24.2 Tris in 800 m1 of water

Adjust pH to 7,5 and Adjust volume to 1 liter. Dispense into aliquots.
Sterilize by autoclaving.
Store at room temperature.
20*SSC.
Dissolve 175,3 g of NaCl and 88,2 g of sodium citrate
~lrto
7~~ops
of JON NaeH.
Adjust volume to 1 liter. Dispense into aliquots .
.sterilize by autoclaving.
Store at room temperature.

in 800 m1 of water

\1.0*PBS;

D1SSoTve' NaCl- 80 g; KCl-2 g; KH2P04-

2 g; Na2HP04-11,34
adjust pH to 7,4. Add 1 ml DEPC and stir for 1 h.
Dispense into aliquots. Sterilize by autoclaving.
Store at room temperature.

g.

III

liter and

1M Tris
Dissolve 121,1 g Tris base in 800 ml of H20DDW,DEPC'Adjust pH to 7,5 with 65
ml of concentrated HCI. Adjust volume to 1 liter.

J")

y;!-'....-

Q(~
Dispense into aliquots. Sterilize
Store at room temperature.

by autoclaving.

~fl1

O,5M EDTA.
Dissolve188,1 .g of disodium ethylene diamine tetra acetate*2H20
in 800 ml
H20DDW,DEPC' Stir vigorously on magnetic stirrer. Adjust the pH to 8,0 with
NaOH (_20 g of NaOH pellets ). Adjust volume to 1 liter.
Dispense into aliquots and sterilize by autociaving.
Store at room temperature.
proteInase

solution.

/~j;;L,i(

i'6f

Mix 10 ml 1M Tris ( pH- 7,5 ), 5 ml 05M EDTA, 35 ml H20DDW,DEPC

andl00
10mg/ml proteinase K.

).1.1

Acetic
anhydride
solution
For 500 ml: dissolve 9.28 g triethanolamine hydrochloride and 4.5 g NaCl in
500 ml H20DDW,DEPC.add 0.25% acetic anhydride (125 l.d/50 ml) just before
incubation.
Washi
lution.
Mix 233,8 g NaCl wit.~ 100 ml 1M Tris (pH-7,5) and 100 ml 0,5M EDTA.
Adjust the vol~me to 1 liter. Sterilize by filtration., Store at RT.
solution
of RNase.
Mix 100 ml RNase ( 10 mg/ml, Sigma
pancreas) with 100 ml 1 * washing solution

R-5503, type I-AS from bovine

(final concentration
10 mg/ml ).