In Situ Hybridization Protocol

7/23/2019 In Situ Hybridization Protocol

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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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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



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

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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.



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



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;!-' ....-



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 ).

In Situ Hybridization Protocol

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