7/23/2019 In Situ Hybridization Protocol http://slidepdf.com/reader/full/in-situ-hybridization-protocol 1/5 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% ethano l- 2 min. 70% ethanol- 2 min. 50% eth ano l- 2 min. \\ ) \ \ \ r ", \ \ \ . ~~ ' , , - ; _ " " " ' 7 ' of the proteins PF A fixative. : , " '\""" - dry. 'S~ Pre hybridization. \ r R inse 5 min . in H20DDW,DEf> C. /~ ~~' \ He at denatu re 30 min. at 70 0 C in 2* S SC. .- : : 7 \.. ~ !...R inse 5 min. in H20DDWD'i P~" - '\~~'\ \ ~ I • " l > • • . • • • - ~-t Pr oteinase K digesti on 1 Q ... !: !!iJ . 370C . ,- - r - ~s, These steps dena tures R NA. and pr obably also removes some ?-~~) . t \ 1 ' \ to make RNA in sections mor e accessible to hy br idizat ion. ,\ Post-fix sp ec imens 20 min. at RT. in freshly prepare d 4% U J ; ; :f . Rinse 5 min. in 0,2% glycine at R T. ~. ~ \_ " '. < S ~ Rinse 10 min. 'n 0.25% acet ic an~ik ~~'-R T' r - ----.----- )) ~ inse 5min. in H20DDW,DEPC. i:l' . \ ' t < , \ = AiT "jdr y specimens , maki ng sure specimens are absolutely \ ' , ' I ., \ \ \ H b . d . t . .. \ .. \ ) \ :;., . A \ " ~ J- \ \ ' u ' : ' : :" \ > ~ y rl Iza Ion ~\\ 0" ' \;' \ ;' ~\" ' - \ ) " V- "" \ ' \, ~,\~~,),\\~\>\. _ l.n . ., , ~ "> \ \ ~ - ~ ~ - ~ -.J - . t r\.i' .\:': ~'rl\'\ ' (. -"-~'<\ \ . . ~':..,,,,,\."'" Solution Hybr idi zati on buff er. 50% Formamid 4*SSCDEPC 1*Denha rd' s 0,5 mg/ml salmon sperm 0,25 mg/ml yest t-R NA 10% dext ran sulfat \ \ \ ,, ' )\ . ' \ Method Mi x the labeled p robe with Hybridizat ion buff er ( estima ting 50-100 III per 22* 22 mm coverslip ) heat to 850C for 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 group of sections and incu bate ~night at 600 in se al ed box con taining paper towels saturated with 5x sSC.~ . -.. .--" ..y ~ ~\~~ \ .' .~ )\ \~1 . \\ · ) \ · 0 . ~ ~ . ~ ~ ~ ~" )\ ' ~ ~ ~ \ , - - - : \ .' \ ' ~ ., J ~ 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 < > " " \ ~
" \ " \ N
-\,~\o J , , , , , - , , , , -
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.].