Double in situ hybridization on mouse embryos for detection of overlapping regions of gene expression

TECHNICAL TIPS 389 389 391 396 397 hrrinda rd zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONM An ine x pe nsive . hom e -m a de zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA & H w be t i Scbumz Doubkinsim ~On-embryosfOr- zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGF ofoverfappfng~ oflpne= prrJaog In S&Jhybridization of whole-mount embryos is an essential tool to study the expression of developmentally regulated genes in mousel and other ver+r?brates.Furthermore, it is often desirable to compare the expression pattern of two d&rent genes in the same embryo distinguishing the co-expression areas (if any) A number of papers have described the use of two in situ probes of various speciesw, using either biotin or fluorescein p&s in combination with digoxigenin probes. The major difficulty in such experiments is to obtaii sensitive and clearIy distinguishable signals during the detection of the two probes. labelled antibodies against digotigenin, fluorescein or biotin, does not usually provide efmugb senThe use of fluorescently sitivity to detect antIsense RNA probes. On the other hand, the use of conventisnal alkaline @~ ~ Phatase or peroxidase sub suates to amplify the signal presents problems when attempting to define accurately the domains of coexpression of two different genes, where the two staining colours overlap. Such areas of overlap normally appear darker than the areas of single staining; they are not visualized as a dlfferent &our, nor as two colours visible at the same time, nor as two &ours visible under different conditions (e.g. under different llhunlnarlons). We describe here a scheme that allows visualization of the expression and co-expression of two genes using whole-mount hybrldizatlon or a combination of whole-mount hybrldlltion and section staining that m a k e s t he areas of co-expression clearly distinguishabk. Our protocol is similar to described procedure@ for hybridization of fluorescein (FIX)- and digoxigenin (DIG)_labeIled RWA probes, which are detected using antibody-alkaline phosphatase conjugates, and combines a lluorochmme with the more usual nitro blue tetrazolium chloride/5-bromo-4-chloro-3-indolyl-phosphate, toruidme salt (NBT/BCIP) staining6. MBT/BClP Gioehringer Mannhelm) are chromogenic substrates and m (Enzyme Labelled Fluorescence mRNA in situ hybridization kit, Molecular Probes) is a fluorescent substrate visible under UV light [a$-diamlno2-phenylindole (DAPI) filter]. FITC-, DIGlabelled and unlabelled nucleotldes, as well as anti-FlTC and anti-DIG antlhodies conlugated to alkaline phosphatase, are from RoelUnger Mannheim. Figure 1 shows a hindliib bud of a El15 d mouse embryo hybridized with a Hardll p&&s (DIG-labelled and RLF stained) and Hox&~ (Ref. 8; PI‘TC-labelled and NBT/UCIP stained) as described below, using transmitted microscope light, W light, and a combination of the two illuminations. Single illuminations show both staining colours alternately, whereas the double illumination shows both staining colour reactions simultaneous!y. allowing proper visualization of the areas where Har d11 ancl Hoxdl 3 are co-expressed. zyxwvuts zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA mc. @J -OBER 1996 VOL. 12 No. IO 385 TECHNICAL The mcthtxlo1o~ zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPON Tms with the modifica- tion5 to the standard protocols a& as follow> (a step-by-step protocol is available 0n rcquc~t). Pcuiflcaelon of probes WA pt.&es xe q’nthesizc.. ustiastanj &mlpmocols and puritied bv pn+itation zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLK ’ with zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA 0 .1 dum t 4 S: lithium &l&e and 3 for 30 min (X2). We do not have any problem with 12-u-l-PDIG (dig~xogc~-lZ-uridine-5’-triphm phate) nuclttiides, but we fmd it is very importxtt to check in a 1% a-r*-TBE gel that unbound 12-UT-F’- : volum~3 ethanol at -20°C FIX nudeor i des hem zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGF a.5 a gr een spot under LV light> do not remain in the probe solution. Unbuund IZ-UTP-FtTC nuclczick?; increase the backgmnd and .so impair the ~&ant pattern signal hybridization UdffrstplVbL? ZZ$IZ!Z we follow the standard procedures on whole-mount mouse embryo+5 hybridizing the two probes si mul t ~ne0usl y. T he main modifications to standard protocols during pre-hybridization and hybridization step are thar we LLLC a I 3 X SSC {pH 5 with citric acid) hybridization buffe r9 instead of the typically 5X SSC. This stringent hybridization sirn~lities the post-hybridiza t ion washing steps. which are reduced to two quick rinses and two washes with the same hyinidi7ation buffer, and a wash with 1: I hybridization buffer:TBST <TrisHCI saline buffer. 0.1% Tween 20, pH 7.6) or MABTfnnlric acid buffer, 0.1% Tween 20. pH 7.5). 30 min arch w a sh a t hybddiia don t rm pe rx ure . We gt? i bener results using FITC-lahell~il prohe for the first developmrnt and incubating with a 1/4ooO-l/8000 dilution of anti-FITC anubody conjugated to Fw;me 1. Double in si#tc hvbridization of mouse embrvo hindliib at 11.5 d. hybridized simukaneously to‘ an antisense Hoxdl .3 p& (FITC-labelled and NBTIECLP stained) and an antin5e Hard21 probe7~* (DIG-lab&d and ELF stained). (a) Whole-mount staining of HoxdU before sectioning.Hard13 has been detected with anti-FITC antibody conjugated to alkaline phosphatase and stained with NBT/BCIP. The Had11 hybridization has not yet been detected with the antiDIG antibody. (b) Har d11 st ai n@ on a 15 w sagittal cryostat section of (aI at the same magnification seen under W light. Har d11 probe, hybridized on wholtmount embryo together with HoxdI3 pr obe. has been detected after sectioning with an anti-DIG antibody conjugated to alkaline phosphatase using ELF as a substrate. (c) Magnification of 6) seen under transmittedlight. Only Hoxdlj hybridization in the mesenchyme can be seen. (d) Same field as (c) seen under W light. Only zyxwvutsrqponmlkjihgfedcbaZYXWV zyxwvutsrqponmlkjihgf Hoxdl l hybridization in the mesenchyme can be seen. Note that the Har d23 hybridization area is smaller than and localized within the distal domain of the Hard1 1 area of expression. le) Same field as (c> and Cd)seen under uansmitted and W light. Note that Hard11 (in green) and Hoxdl.3 Cm blue-purple) can be seen at the same time in the area of co-expression. and Hard11 can be also seen in the rest of its expression domain (green-yellow). Reciprocal in SUU hybridization in the opposite hindlims bud of the same embryo (not shown) shows the same pattern of expression and co-expr&on but with the staining colours exchanged. Bars: zyxwvutsrqponmlkjihgfedcbaZ (a. bP 0.4 mm; (c-e)= alkaline phosphatase. 0.1 mm. The fi probe has to be developed with NBVBCIP substrate. Emhyos older than 8.5 d should be .sectioned to allow detection of the fluorescent substrate. Appropriate sections can then be c hose n fGr detection of the second probe in ams of expected co-expression. We typical$ do so after taking pictures of the whole-mount staining. DC not re-foe the embwos; cryoprotect in 30% sucrose and embed in OCT (Tissue Tek OCT compound, Miles). After sectioning. detection of the fti probe by NBT/BCIP staining can be re-started again if the tkt prc#e staining is not visible enough by washing in staining reaction buffer (So rnin) and adding fresh iVBT/E+CIPsubstrate. When developed to the desired extent, kill the activity of the first phosphatase by incubatmg either with glycine-HCI buffer pH 2.2 (for 1 hl or methanol (Zr: 15 min). both work well. Incubate with the anti-DIG antibody (l/2000-1/8ooo), wash extensively with TEST or hMBT and then prwced according to the instructions given by ELF supplier. Generally KM-150 ml of ELF working substrate per slide, kept in rl humidified c ha m be r in t he da rk . is e nough. Good second colour reaction is usually obtained in 1-24 h. PeriodicAlly check the extent of the colour reaction under Uv light usi?g DAPl filter.Wash for 15 min. Do not wash extensively after the mlour reaction as this cxn remove that fluorescent preapltate. The sections c;n be counterstained with Hoechst 3342, DAPl or pmpidium lodldc (Hoechst 33342 is provided in the ELF staining kit). For long-term storage, post-fut the staining with 2% formaldehyde Xl mg ml-t MA in phosphate-buffered saline PBS for 30 min and mount in Citifluor or ELFmounting medium. lf. after the fint probe development, sedioning is required !typially for embryos dder than 8.5-9.5d), it is useful to pefiom ;I duubk i#rs&r hybridimtion and its reciprocal, in order to obtain whole mount and overlapping pattern images of both analysec g~nea. Thr \kuali;llttiun uf the expression and co-expression of two different genes that this methodology provides allows ccwxprehsion domains to be defined accumtely (Fig. 1). The method does not, however, allow co-expression to be identifiec within an individual cell due to the nature of the ELF substnte precipitate, which form crystals instead of a homogeneous stain 1°Kprcclpitate. 1’he dark blu<bpurple NBT/BCIP precipitate c%nmask weak Eti staining (e.g. when using to detect weak probes) hur we have not found this to be a problem with our probes. If masking should occur, we suggest using the N5T/ElCIP to detec the wc-aker prolx Finally. we have obtained the same results performing the double in sitar hybridmtion on cryostat as well a! on paraffin scaions WC have followed the standard protocols for in si t u hybridization on sections’” with the modifications statec aln)vc. Thcw modifications wil! prob_Jbly prove applicable IO a wide variety of experimental systems. TIG OCTOBER 1996 VOL. 12 No. 10 386 TECHNICAL TIPS zyxwvutsrqponmlkjihgfedcbaZYXW zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIH fhrorescent frr situ hybridizationro zyxwvutsrqponmlkjihgfedcbaZYXWVU zebrafish embryos networks that reguiate devrlopment. rt i\ ofp:n zyxwvutsrqponmlkjihgfedcbaZYXWV use ful 10 know whether individual cells XC’ \lmul- Double To undcrsrand the genetic aneously expressing more zolour in situ hybridization than one gene ~zpcctcd 10 be invoh cd in J particul;lr iJr<,cck\. This que%hcm an be 4vert hy t~\r* using chmmogrnic suh>tr.ltes’.l in ti\\uc sealc,ns or ~1hole-mount e m b ryo % . If the domxns of this p rc hlc m b y the stxining o f o ne c hro m o g e n a n m a 4 rlxtrd ~htorher. We have overcome using fluo re sc e nt sub stra te s fo r a lka line p ho sp llntnse - c c )nj~l~te d antilxxlies WC have rested rhrclr difTert,nt Fast Red w hsmtc.s: t 3 (V e T or La hor~l~~ri~sI . (1 1 f,M Ikd tahlera~ (Raehringer .Clannhclm). and C1) Vectorm Red alkaline phosphatase subsrr~c 13) Sigma Fa@’ Fast Red TR/ Na p htho l A S- .LiX < Sig m a ). Bc h < uh< tr.rle ha ke e n WC C I in c o m hin:ltio n with the c nzym c la tx4c d fluo re sc e nc e sub stra e (ELF’. Mo le c ula r Pro b e s). O ne p ro b e is la !+ 4!c d with tluorcs~?in. and the other with digo:ouigenin. e nd they are visualized hy srqurntial inculrJtion in alkaline phosphJta.w4 nnlugarccl anubcxlies to ffunrr.xrin and digr>xigenin. rcspectively. The first antibody is staned with one of the three Fa.51 Keti formulatlon~ and the second with rhe ELF 5uhsrnrc The 1.(,-di:lminn-2-ph~nylindole) lilter ~‘t.5. re spc c tike ly signals a re vie we d b y fluo re sc e nc e m ic ro sc o py with rho d a m ine an’1 DAPI i% ~o le c ub r Pro b e s sup p he s :I va ie ty o f k1t.s c o nta ining ELF T” 51 h:;irXe . WC ilx the !4 iht l3 t f? h~miulatton in the ELF- hP immunohistochemistry kit (E-M’100). mxle up in Tris-HCI ijuffer at pfl 8.2. VrcrmrTw Red i.c The different Fast Red suh\tmtcs hzve d&rent chardaenstics. m a d e up from three solutions provided in the kit. St.linmg is quite r~pld. appc:mng within a few minutes to l-1 IX Prolonged incubation tends not to inter&y the signal. With zehr&sh cmhryos. the yolk ztainb ~~lknv hut background m rhc embryo rcmainb lo w. The Fa t Red from &whring e r Ma nnhe im is in the fo rm o f k1hle t.s. The srrlut~ m p ro d uc ts 3 pre c ipita te a fte r p rrrlo ng c d I~C LIb a tio n. The signal de ve lo ps !e szzquickly than Vecto? Red. hut producrs :I mart’ intense red precipitate. B;tckgounds can TV q uite o ra ng e in the yo lk a nd in the e m hvo . With Sig m a FC JSF’ Fa st Re d . ta hlc ts a rc ‘ ~ p p p fie d fo r b o th the Td s b uffe r a nd the Fat Red substrate. The intensity of the signal and the speed of development is slmllar tcr Hochriilger FJ .s~ ‘~ Re d. l%ckgmunrl% are 3 Red AX> produce5 le.%\ little less than with the Roehringer Fnht T’ I Red hut greater than with Vccrt)r*” Red. %gma FM T’I AI three .suhstrJteh .fre less sensitive th:m the mrosf frequently precipitate in the staining solution than the Btwhrin,+ 11-r suhstrztr. used substrate combination of nitro blue tetnzolium chlundei5-hrom~-chlort~3-rn~lolyl-phosph;lt~. toluidme-salt tNUT/BCI?. Boehringer Mannheim). However, we have detected s1gnal.s wirh a Iarge numhr of diffcrenr zebmfish probe.<. With the R.CA+X fluorescence becomes 3 problem. For strung prolxa\ t11c*hcst hignal-to-noise rztira arc ohrzined wirh signals the background Vector’” Red. The alkaline phosph;ltare an he inactivated by hc~t treating dt hi°C for 30 min \\i!h the V&o? Red precipitate. bui bofh of the other Fast Red precipitates 3re lost If heat treated. Therefore. the ;rlkalme phc>\phatase actlviry ir; inactivated hv incuharinp in 100 rnbl glycine pH 2.2. 0.1% Tween-20 for 30 min. i?xce&ve .stSning \\iith the Fat Red .should IX avoided beau.sc :I bar-y red precipitate an panially quench the Ew” signal. We always perform the ELF staining 35 the second rtzraion 3s It 1~ more .senhlti\c rllan the Fast Red 4>~tr~tc~ and can be left to develop for up to 24 h. The inactivation step is VCT): imponant 3s falure to complercly inactivate the fint alkaline phrrsphsa*e will g ive 3 we a k sig na l with rhc ELF. which appears to be co-ltx~lized with the t:Jst Red precipitatr. Thus. we \ug_gcst perforaiing the fo llo wing c o ntro ls. For some embryos. in place of incubation in the .scconcl antibody (-rep 3 below). cc:ntinne to incubate in blocking solution for the same period and then ctxxmur w~rh the W:!shc\ a though antiixxty had lwen added. Suh.sequen~ sta ining with ELF sho uld b e ne g a tive o r g ive a n e vrn, no n- lo a liz~ d ha c kg ro und . (1) Perfo.m reciproccal q wrim e nts in whic h A, l&!led with Huo rc we m a nti vi.wa lizd with Fa t Re d : p ro k the visua lia tio n m e tho d s fo r the p ro b e s a re re vwe d : (i! probe R, labelted with digoxigentn and visualized wrh ELF: (ii) p ro b e IS. la b e lie d with tluo rc sc e in a nd visualized with Fast Ked; probe ELF signal 15 rmly detected for one probe when FJ 3 .r Red is A, labelled with digoxigenin and visualized with ELF If ctrltxdircd used to de te c t the strongest signa! then the inactivation of the alkaline phn@ar;lx~ 1~1s probably incomplere. (3) H~hndizitions should be carried out with single p ro b e s a nd \&u&&on with fisr Red. ELF and V+T/BCIP sepxxrely to dertxnime direshalth of detection for different domains of e xp re ssIo n o f the tm nsc np t. - xp re ssio n 2 o ve rla p . ho we ve r. zyxwvutsrqponmlkj C LII are Fast (1) 387