in situ hybridization protocol
TRANSCRIPT
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Depar a ffini zation and r ehydration of t he s pecimens
(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.
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of the proteins
PFA fixative.
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d r y. 'S~
Pre hybridization.\ r R inse 5 min. in H20DDW,DEf>C.
/~ ~~' \ Heat denature 30 min. at 700C in 2* S S C. .- : :7
\..~ !...R inse5 min. in H20DDW D'iP~"- '\~~'\\ ~ I • " l > • • . • • •
- ~-t Pr oteinase K d igestion 1 Q ... ! :! ! i J . 370C . ,- - r
- ~s, These steps denatures R NA. and pr obably also r emoves some
?-~~) . t \ 1 ' \ to make RNA in sections mor e accessible to hy br id ization.
, \ Post-fix specimens 20 min. at RT. in freshly prepared 4%
U J ; ; : f . Rinse 5 min. in 0,2% glycine at R T. ~. ~ \_
" '. < S ~ Rinse 10 min. 'n 0.25% acetic an~ik ~~'-R T'r - ----.-----
)) ~ inse 5min. in H20DDW,DEPC.
i:l' . \ ' t < , \ = AiT" jdr y specimens, making sur e specimens ar e a bsolutely\ ',' I
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SolutionHybr idization buff er .
50% Formamid
4*SSCDEPC
1*Denhar d' s
0,5 mg/ml salmon s perm
0,25 mg/ml yest t-R NA
10% dextr an sulfat
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Method
Mix the labeled probe with Hy br id ization buff er ( estimating 50-100
III per 22* 22 mm cover sli p ) heat to 850C f or 5 > min and coolon ice.
Take slides after air-drying and pipette 100 ~ lover each gr ou p of
sections. Gently put parafilm over each gr oup of sections and incu bate
~night at 600 in sealed box containing paper towels saturated with5x sSC.~ . -.. .--"
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In situ hybridization Protocol
For paraffin sections
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Washing a f ter the hybridization.1. Wash the slides in 5* sse until the coverslips were slide off ..
2. Wash in.2*SSC +50% f ormamide at 650 e f or 30 min, 300 ml f or 10-20
slid es.3.2*SSC 3 times f or 5 min, t 37 e~.~:~ashin,,"g solution + . 1 ~/ml RNAz~, t 37 C, 30 min..,..•.. -.J~C I:JD~f urmamide l~ , . , _ n
6.2*SSC t 37 e 1-5--m iR ~\ '" ~ -
t- t~"f -. 7.~SC t37 C 15 min
1 < > " " \ ~
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I mmunological det ect ion of DIG-labeled hybr id sWash in\TBS 3 times f or 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 R T.~; ~\\~'" ~
R eplace the blocking solution by: alkaline phosphatase conjugated anti-DIG
antibod y d iluted 1:500 in 1% goat serum ':CBS f or 60 min at R T.
Wash 5-10 times in TBS f or 30 min.--)?::~~"e:\~ ! ( Q \ ~ ~ r \ - ~\l,I<~" i > -
To per f or m th~ col9.~~r eaction: add '''', 0 ~l NBT, j i f .5 ~l X-phosphate solution
and 2.4 mg le~isole to 10 ml buffer containing O.IM Tris pH 9.5, 0.05M . ; JMgC12 and O.lM NaCl. 4-~\ DOW1 o·S •.•.•\ Tv-;<" A ~. B.( 1~'O.w f .$-\ 11 M : " < J , . f
\00M- ~ N • .•.,f ~M
Incubate in RT f or f ew hours or in 40 e f or on or few' days, d e pend on the
background and signal.
Pr e parat ion of RNA probe for in sit u hybridization
A. R estr iction d igest and purif ication of the plasmid.
For anti-sense pro bes (which hybrid ize to the RNA), linear ize the plasmid
containing the cDNA insert by cutting it with a r estr iction enzyme at the
or iginal 5 -end of the cDN A insert (or outsid e at the poly linker). Use the
_ polymerase going fr om the or iginal 3-end of the insert towards its 5-end .
Use the same logic f or the sense probes (Le., cut at the sid e of the 3-end of
the insert and use the R NA polymer ase going toward s it).
a. Extr acting once with phenol-chlorof orm (i.e., add an equal volume of
Tris equilibr ated phenol-chloroform mixture, vortex for 30 seconds spin
for 5 min. and take the UPPER aqueous phase f or step Itblt).
b. Ethanol precipitation [add sod ium 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 f or 2 min.Spin f or 10 min.].
c. Wash the pellet with 75% ethanol.
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d. Re suspend in H20 to yield 1 mg/ml.
e. Ver if y 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 "BRLft
). For
detection do not load more than 0,5 mg of the DNA and 1 mg of the mar k er.
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 buf f er
50% v/v Glycerol
0,2M EDTA pH 8,0
0,05% w/v Br omphenol Blue
B. The LabelingAdd the following items in the order they appear : (note,
should be RIBOnucleotides and NOT the deoxy f orm)
1. D NA( approx. 0,5 Jlg to 1 Jlg)
2. 5* transcription buffer ( arr ives with the enzyme)3. Dig R NA Labeling mix, 10*conc.
4. DTT (100 r ru\t1 )
5. RNase inhibitor( like RNasin )
. 6, RNA polymerase(TI/TI/SP6 )
7. Water
Incu bate 2h 37 C.
Next, add to the reaction the f ollowing:
1. RNase inhibitor
2. DNase
Incubate 10 min. at 370C
1 III
2,5 III2,5 ). L l
2,5 III
1III
1 III
14,5 III
Following this incubation add :
I.EDTA 0,5M ph 8.0 1,25 Jl1
2.LiC14M 3 ,1111
3.Ethanol( -20 C} 9 0 1 1 1
4. Mix well and store at -20 C for 2h or over night.
5. Centr iguge at 12000g at 4 C for 15 min.
6. Wash the pellet with 70% ethanol
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Reagents and solutions
4 % paraf ormaldehyd e in PES.
To prepar e 100 ml of fixative:
Add 4 g of par af ormaldehyde to 50 ml water .
Ad d 6-10 ~l of 10 M NaOH.
Ad d 10 ml of 10*PBS.
Adjust the volume to 100 ml with water .
Stir at 650C untill the solution will become clear .
Stor e at 40C f or up to 2 4 h.
H20DEPC
Add 500 ~l of Diethyl Pyr ocar bonate (DEPC, SIGMA, D- 5758) to 500 ml
H20DDW'
Incu bate flask with H20DDW.DEPCin water bath at 370C f or overnight.
Sterilize by autoclaving.
Store at r oom temper ature .~.
All the solutions which used f or "I N 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 Ad just volume· to 1 liter. Dispense into aliquots.
Ster ilize by autoclaving.Store at r oom tem peratur e.
20*SSC.
Dissolve 175,3 g of NaCl and 88,2 g of sodium citrate in 800 m1 of water
~lrto 7~~ops of JON NaeH.
Ad just volume to 1 liter . Dispense into aliquots .
.sterilize· by autoclaving.
Store at r oom temperature.
\1.0*PBS;
D 1 S S o T v e ' NaCl- 80 g; KCl-2 g; KH2P04- 2 g; Na2HP04-11,34 g. III liter and
ad just pH to 7,4. Add 1 ml DEPC and stir for 1 h.
Dispense into aliquots. Ster ilize by autoclaving.
Store at r oom temperature.
1M Tr is
Dissolve 121,1 g Tr is base in 800 ml of H20DDW,DEPC'Ad just pH to 7,5 with 65
ml of concentrated HCI. Adjust v olume to 1 liter .
f I )
J " ) y;!-' ....-
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Q(~~f l1Dis pense into aliquots. Ster ilize by autoclaving.
Stor e at r oom temper ature.
O,5M EDTA.Dissolve188,1 .g of d isod ium ethylene d iamine tetra acetate*2H20 in 800 ml
H20DDW,DEPC' Stir vigorously on magnetic stirrer . Ad just the pH to· 8,0 with
NaOH ( _ 20 g o f NaOH pellets ). Adjust volume to 1 liter.
Dispense into aliquots and ster ilize by autociaving.
Store at room tem per ature.
proteInase K solution. /~j;;L,i( i'6f Mix 10 ml 1M Tr is ( pH- 7,5 ), 5 ml 05M EDTA, 35 ml H20DDW,DEPCand l00 ) . 1 . 1
10mg/ml pr oteinase K .
Acetic anhydr ide
For 500 ml: dissolve
500 ml H20DDW,DEPC.
incu bation.
solution
9.2 8 g tr iethanolamine hyd r ochlor id e and 4.5 g NaCl in
add 0.25% acetic anhydrid e (125 l.d/50 ml) just before
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 f iltr ation., Stor e at RT.
solution of R Nase.
Mix 100 ml R Nase ( 10 mg/ml, Sigma R -5503, type I-AS f rom bovine
pancreas) with 100 ml 1* washing solution (final concentration 10 mg/ml ).