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Detection of mitotic spindles in third-instar imaginal discs of Drosophila melanogaster
TECHNICAL
I 5 6 I 7 8 j 9 10
TIPS
Hall, J..'~l. et d. (1992) Am.J. Hum. Gener. 50, ]235-1242 Kwi,atkov,'ski, DJ. and Diaz, M.O. (1992) Hum. Mol. Genet. 1, 658 Vanagaite. L. (1994) Genomics22...231-233 Zhang, L., Leeflang, E.P., Yu,J. and A.q'dleim, N. (1~)4) h2tt. Ge~et. 7, 531 Urquharh A., KJmpton, C.P., Dowries, TO. and Gill, P. (19°A) Iltr.]. LegalMed. 107,13-20 Hazan, J, etal.(1992) Genomicsl2,183-189
I
Contributed I~, Fn;ddric Fottcault, Franfoice Praz, Cbrislla~t Jaulin attd Mou~#a Amor-Gueret* (iocmb~clubqnter~letfr), ] lahoratoile tit. 3lutag6r£~ et Patl)ologie Humaine', h~stgut Jacques Monal, CNRS UMR 9922, Tour 43, 2 Place Jttsstett, [ 7{ 251 Paris cedex 05, France. Present address: Laboratoi~ de 'G~16tique Mol6culaire et Canc~rogdndse'. ]OCMH, [ 1 2 9 R°ute d~"Stali'l~ra'/' 93000 B°big"y" rra:ge~ " [ .
.
.
.
.
.
.
.
.
.
.
.
I~ec~on of mitotic spindles in third-instar ima~nal discs ofDrosopb#a metanogasler
£,
Whole mlaLot immunodetection techniques ;Ire commonly used in Dmtso~Ala melanogasler et'nlyi',/os to :itudy the cell cTcI~ Ity vL~ualizatinn of mitotic spindles. However, sometimes it ks necessary to dt.¢ecl and ~udy mitotic figures at larval stages. Tbe most available diploid larval tissues ;ire bKtin and imaginal di~'s. In most of these timues, cells divide throughoul laP'a] life. Up to now, larval mitotic figures have only been recorded from brain. Immunodetecgon of mitotic spindles in imaginal discs has proved difficult because both the structure of mitotic spindles and the integrity of imaginal discs must be preserved. Mitotic spindles are vety lal)ile struc'tures. Mk'rotubules are known to readily depolymedze under moderate [L~ttire conditions such as the low cx~ncentrations of formaldehyde (3,'?'/0 or 4%) commonly used in prottg:ols for whole-mount immut'~ostaining of imaginal d L¢,CS.One of those protocolst allows the conservatinn of some mitotic spindles hot not in a reproducible manner. I NeiSer do the conditions described to prepare and fix larval brainsZ (dissection in 02% NaCI and fixation in 0.7~'/0NaCl, 3,~b formaldehyde) allow the conservation of mitotic spitxgt.~: in imaginal discs, "[11¢harsh conditions u ~ d to fix embryos ~(MeOIt at -20~C or 37~0 Ibrmaldehyde) cause damage to imaginal dims. To ,solve these problems, we have modified the classical techniques of immunodetection. Critical ~eps a ~ di~sectinn, fixation and penneabilization d tissues. Numerous fLxativeconditions were tested by changing the fixative agent anti its concentr,uinn, tile chelator agent concentration, and the pH of the gxalive solution. The dissediun and perrneahifi;,;ttinn steps must be controlled to avoid alt~..ratinnof discs and to obhqin homogenous staining. 'l'l~ mitotic ngurt~, in imaginal discs were lalrelled with fluore.,;cen~ antilxalies and vlsualiznd using contbcal microscopy. The seoal sections permit a three-dlmensinnal study of the relationships Ixtween components of the mitotic cells. We have devised a method that conserves mitotic spindles without the addition of a stabilizitxg agent which t~an induce artefact.,;. "l'his method allows both tile visualiz~tinn of spindles during all m!totic pbases and tile visuali;,.ation of interpb'asie mit'rotuhnlt.s (Fig, l ). l"hes¢~conditions can also I x used in double malning ~o detect other mitotic ~lmcturL*ssuch as Cenlrosomes or ehromosoules. ,
P r e p a r a t i o n of t h t d . h ] s t a r lmaginal discs
I ~)Lsse~hrva~discsin~h¢~pba~e~bu~redsa~ine(~[k%~6~t~jH2N~6n~`~t~`~HNa2` 0.15t4 NaCI, pH 7.51. In,.,elt larvae to free the dk'd. It is crucial fllat this step is t~rded out rapidly at n)om temperature as chgling on ice eventually disassembles nlicfotubules. FLxatinn must quickly follow dissection. 2 Fix discs in I ~ fresh forrmlklebyde t Fluka), I mM EGTA, PBS at room tempe~ture for 20 min, The fixative solution must Ix- :idjtlsled to p| [7.5 witb die solution, 1.4 ',x,I. 1 M NaOH, I voL 1 M KOI L This high cont'entration of fommldebyde allows a rapid fix* atinn of microtubules organized in spindles and does t:ot produce broken discs ,as dtves ftxalion with Mt~OII at -20°(; or 37% fomraldehyde. Our mode of fLx,qtion results in v,'ell-.stmcturnd spindles such as those that we observe when the microtubule stabifizing agent taxol (0.5 m~,t)is indoded in tile tLxative soltuion. The prc.K'edure without raxol was prefen'cd to limit artelk'ts. Flatlml !. Mitotic figures of dlird-insrar eye-:lntennal imaginal discs of Drosopb#a melanog~ter. (a) ct-Tubulin staining of a tell in anaphase at high magnifit~ation.The spindle morphol%,y is well prt~erved and individual microtabules can be observed, (b), (O Duuble-anlihndy staining revealing u-tubulin and centmsnlnes. Ib) u-Tubulin staining showing a midlxxly characteri.stic of late tclopha~ (arrow) and the netvork of interphasic microtubules in surrounding cells. (c) Centrtx~rme staining using antilx',dy Rh188. 1111s antibody recognizes a protein that is nttclrar during interph:t,se and centro~mal dudng mitosis. C.entrosomes ate clearly visible i in file daughler cells (arm;vs), lmll~unnfluures~,ence micaxscopy "¢.-a.* c'arrit.'d out using a Mgc 609 BIo-Rad contkra[ microscope. TIG NOVEMBER1996 VOL. 12 NO. l 1
452
TECHNICAL
TIPS
3 Rinse twice in PBS and w ~ h once in PBS for at least 5rain. 4 Penlaeabilize discs in 0.3% Twecn, PBS for 10 mill. Penneabilizing lbr longer c:m :liter the dlsx's, :is does pemaeahilizaflon with MeOH. 5 P~incubate discs in pitT (0.1% Twc~n, pIIS), I).1% BSA for 30rain to 1 ll.
Antllme. staim~ 1 Incubate discs with pdnxary antibodies in PBT, 0.1% BSA, 2/6 norutal serum at 4°C, ovemight (normal serum is lioru tile species used to raise the secondary antibody; if two secondary antil~xlics art., net.dud, IL'~e1% nornxd serum from cIch apecies). To detect tr~totlc spindles, we used a tlionoclunal anlilX:dy raised in mcmse against chick et4uhulln (Sigma~ T-9026), at a dilution of 1/200. Oentaosomes can he visualized using the lx31ycinnal antilaxly Rb188 raised in mhhlt against a Drosophila centrosomal protei#, diluting the arui,~rull'l 1/200, 2 Wash 4 - 6 times in PBT over a 1 h period, Finally, wash in PBT. 0,1% BSA. 2% normal serum for 1 h. 3 lncubale with secondary antibodies in PBT. 0.1% BSA. ~,6 nonnal serum for 211 at ix)ore temperature. Secondary antibodies are conjugated with fllodamine or fluorescein. Dilutions depend on tile secondary antilmdy used, e.g. mcondary antiixfly a~,linst rabbg [gG(H+L) ¢oniu.qatod widl dloc'lanline (Biosys. Bi22flT), 1 : 50, secondary aNtil'~My against muusv 1F,,G(H+L) conjugated with H I E flnsgtut Pa.steur Ptod~actlon), 1 : 200. To reduce background, secondary antilxxlles can Ix' prc-abscdxxl on larval heads dissected, fixed and pemleabilized m'~in tile experiment, Pre-absorh by diluting tile ,~coodary ;mtilxxty 1:8 ha t h r ~ volumes of FBT. 0.1% I~;A, 2% normal senlnl against one volmne o[ fixed ti,~sues, for 2 h :it lX~olnlemperatulx', ,t Wash ixvice in I)BT and e.vice in FBS over a 1 h period. If DNA staining is desired, incubate fls,sues wifl'~the appropriate rtnlgent I~fotc tile PIK";washes. We u.~od 0.5 nlg rul-I dlix)momycin (Sigma C-2659) in PBT for I h at 37°C, which can Ix: detecttxi by confocal imag ng. For convent onal fluores¢cmce i*l cro.scopy, use 0.5-0.1 illgm1-1 DAI , I%PBT for 30 Ill n to 1 h at room ienlpemture. Then, ;~msh P,vice in PBT and twi..'e in PBS o'¢er a I It period. 5 Mount tilt' preparation in Citifluor (Citifluor Lid, London). Discs must he well Ilattencxt to allow obsela,ation. During tile whole process, discs are manlpulmed in glas~s cupels. V(lhnnt.~ art 400 ILl lot washes and 200 ILl for antilx,xly incubations for 10-15 larval heads. Gentle agimtinn is used during tile difl~ercnt int'ubagon and rinse steps. After addition ¢ff fluorescent antilx',dies, incuhatinns are made in tile dark add manipulation can 1~2carried out under a dim grt',2n light, ACKNO~t:U:I)GEMEN'fS We we'.lid like to thank William %~ritfield f¢lr proGding us with Ihe flb188 amilxvdy. This work was supporled by tile Cemre National de la Redlerche Scientiflque tUMR 9922) aod by tile Unive~,~itiesIL aml M, Curie aml D, Did¢~at. REFEBERCI:~ I Stoahl, G. and Basler, K. (1993) Cell7!, 527-540 2 Gonzal~z. C. and Glover, D.M (19931 in T/)e Cell Cyde, A PracticafApp)~lcb (Fmntes, P. and Brtmks, If,, ed.s), pp. 143-175, IRL Press 3 Theurkauf, W,E, t 1992) Dt,v. BioL 154, 20~-2t7 4 Whltfleld, W,G.F, el aL (1988).L c't.I/Sct 89. 467-.180
Cotm*lmtt, d I~l>A,tp~flsAitdif~9"l faltdilvert@cer~u.<~ie~,~.), Altti)t FA,IlvCtold Ma~lfllv Sfl)tottt.liR, D i'uamique du G6nome el Etxdutio., htstitut .[aceplt,$ Moilod, tl~iit~9~it6 Dt.tlL~ Diderot, 2 place Jtt.~ietl, 75005 Parle. arid ~G')olll~' de qt;tldtiqlte Celhdaim et Moh;culaim lIRA CNR$ I L~5 '13iologieMok~ctdaJre el Ck,lhdaO~, d . lX~t~'loplx'me~it" U)lfl%.l~llt~Pfe~. d Marie Clirlt'. 7 ql~ai,Qdlll Bvmatr~ 75005Paris, Fro)ice,
R a p i d PCR-medlated b i d i r e c t i o n a l d e l e t i o n s
-
-
-
51nc~ tile introductkm of PCR, great eltbrt has I~en put into ino~asing the size of its products t. Every new PCR plxRJu~.l must be identified, and ~quencing iX#lll:linstile m a t impotlant nleglod of chamctelizatiun. After t )verconling initial dilllculti~, direct sequencing of FCg ploducLs was eslablisJled as a method of dloi¢,2, Pdther than s~quun¢ing of CIollc~i PCR produc'ts, Inainly ]~cau~¢ tri" thv wcll.
TIG NOVI-~MBER1996 Vt)L. 12 No. 11
453
I 5 6 I 7 8 j 9 10
TIPS
Hall, J..'~l. et d. (1992) Am.J. Hum. Gener. 50, ]235-1242 Kwi,atkov,'ski, DJ. and Diaz, M.O. (1992) Hum. Mol. Genet. 1, 658 Vanagaite. L. (1994) Genomics22...231-233 Zhang, L., Leeflang, E.P., Yu,J. and A.q'dleim, N. (1~)4) h2tt. Ge~et. 7, 531 Urquharh A., KJmpton, C.P., Dowries, TO. and Gill, P. (19°A) Iltr.]. LegalMed. 107,13-20 Hazan, J, etal.(1992) Genomicsl2,183-189
I
Contributed I~, Fn;ddric Fottcault, Franfoice Praz, Cbrislla~t Jaulin attd Mou~#a Amor-Gueret* (iocmb~clubqnter~letfr), ] lahoratoile tit. 3lutag6r£~ et Patl)ologie Humaine', h~stgut Jacques Monal, CNRS UMR 9922, Tour 43, 2 Place Jttsstett, [ 7{ 251 Paris cedex 05, France. Present address: Laboratoi~ de 'G~16tique Mol6culaire et Canc~rogdndse'. ]OCMH, [ 1 2 9 R°ute d~"Stali'l~ra'/' 93000 B°big"y" rra:ge~ " [ .
.
.
.
.
.
.
.
.
.
.
.
I~ec~on of mitotic spindles in third-instar ima~nal discs ofDrosopb#a metanogasler
£,
Whole mlaLot immunodetection techniques ;Ire commonly used in Dmtso~Ala melanogasler et'nlyi',/os to :itudy the cell cTcI~ Ity vL~ualizatinn of mitotic spindles. However, sometimes it ks necessary to dt.¢ecl and ~udy mitotic figures at larval stages. Tbe most available diploid larval tissues ;ire bKtin and imaginal di~'s. In most of these timues, cells divide throughoul laP'a] life. Up to now, larval mitotic figures have only been recorded from brain. Immunodetecgon of mitotic spindles in imaginal discs has proved difficult because both the structure of mitotic spindles and the integrity of imaginal discs must be preserved. Mitotic spindles are vety lal)ile struc'tures. Mk'rotubules are known to readily depolymedze under moderate [L~ttire conditions such as the low cx~ncentrations of formaldehyde (3,'?'/0 or 4%) commonly used in prottg:ols for whole-mount immut'~ostaining of imaginal d L¢,CS.One of those protocolst allows the conservatinn of some mitotic spindles hot not in a reproducible manner. I NeiSer do the conditions described to prepare and fix larval brainsZ (dissection in 02% NaCI and fixation in 0.7~'/0NaCl, 3,~b formaldehyde) allow the conservation of mitotic spitxgt.~: in imaginal discs, "[11¢harsh conditions u ~ d to fix embryos ~(MeOIt at -20~C or 37~0 Ibrmaldehyde) cause damage to imaginal dims. To ,solve these problems, we have modified the classical techniques of immunodetection. Critical ~eps a ~ di~sectinn, fixation and penneabilization d tissues. Numerous fLxativeconditions were tested by changing the fixative agent anti its concentr,uinn, tile chelator agent concentration, and the pH of the gxalive solution. The dissediun and perrneahifi;,;ttinn steps must be controlled to avoid alt~..ratinnof discs and to obhqin homogenous staining. 'l'l~ mitotic ngurt~, in imaginal discs were lalrelled with fluore.,;cen~ antilxalies and vlsualiznd using contbcal microscopy. The seoal sections permit a three-dlmensinnal study of the relationships Ixtween components of the mitotic cells. We have devised a method that conserves mitotic spindles without the addition of a stabilizitxg agent which t~an induce artefact.,;. "l'his method allows both tile visualiz~tinn of spindles during all m!totic pbases and tile visuali;,.ation of interpb'asie mit'rotuhnlt.s (Fig, l ). l"hes¢~conditions can also I x used in double malning ~o detect other mitotic ~lmcturL*ssuch as Cenlrosomes or ehromosoules. ,
P r e p a r a t i o n of t h t d . h ] s t a r lmaginal discs
I ~)Lsse~hrva~discsin~h¢~pba~e~bu~redsa~ine(~[k%~6~t~jH2N~6n~`~t~`~HNa2` 0.15t4 NaCI, pH 7.51. In,.,elt larvae to free the dk'd. It is crucial fllat this step is t~rded out rapidly at n)om temperature as chgling on ice eventually disassembles nlicfotubules. FLxatinn must quickly follow dissection. 2 Fix discs in I ~ fresh forrmlklebyde t Fluka), I mM EGTA, PBS at room tempe~ture for 20 min, The fixative solution must Ix- :idjtlsled to p| [7.5 witb die solution, 1.4 ',x,I. 1 M NaOH, I voL 1 M KOI L This high cont'entration of fommldebyde allows a rapid fix* atinn of microtubules organized in spindles and does t:ot produce broken discs ,as dtves ftxalion with Mt~OII at -20°(; or 37% fomraldehyde. Our mode of fLx,qtion results in v,'ell-.stmcturnd spindles such as those that we observe when the microtubule stabifizing agent taxol (0.5 m~,t)is indoded in tile tLxative soltuion. The prc.K'edure without raxol was prefen'cd to limit artelk'ts. Flatlml !. Mitotic figures of dlird-insrar eye-:lntennal imaginal discs of Drosopb#a melanog~ter. (a) ct-Tubulin staining of a tell in anaphase at high magnifit~ation.The spindle morphol%,y is well prt~erved and individual microtabules can be observed, (b), (O Duuble-anlihndy staining revealing u-tubulin and centmsnlnes. Ib) u-Tubulin staining showing a midlxxly characteri.stic of late tclopha~ (arrow) and the netvork of interphasic microtubules in surrounding cells. (c) Centrtx~rme staining using antilx',dy Rh188. 1111s antibody recognizes a protein that is nttclrar during interph:t,se and centro~mal dudng mitosis. C.entrosomes ate clearly visible i in file daughler cells (arm;vs), lmll~unnfluures~,ence micaxscopy "¢.-a.* c'arrit.'d out using a Mgc 609 BIo-Rad contkra[ microscope. TIG NOVEMBER1996 VOL. 12 NO. l 1
452
TECHNICAL
TIPS
3 Rinse twice in PBS and w ~ h once in PBS for at least 5rain. 4 Penlaeabilize discs in 0.3% Twecn, PBS for 10 mill. Penneabilizing lbr longer c:m :liter the dlsx's, :is does pemaeahilizaflon with MeOH. 5 P~incubate discs in pitT (0.1% Twc~n, pIIS), I).1% BSA for 30rain to 1 ll.
Antllme. staim~ 1 Incubate discs with pdnxary antibodies in PBT, 0.1% BSA, 2/6 norutal serum at 4°C, ovemight (normal serum is lioru tile species used to raise the secondary antibody; if two secondary antil~xlics art., net.dud, IL'~e1% nornxd serum from cIch apecies). To detect tr~totlc spindles, we used a tlionoclunal anlilX:dy raised in mcmse against chick et4uhulln (Sigma~ T-9026), at a dilution of 1/200. Oentaosomes can he visualized using the lx31ycinnal antilaxly Rb188 raised in mhhlt against a Drosophila centrosomal protei#, diluting the arui,~rull'l 1/200, 2 Wash 4 - 6 times in PBT over a 1 h period, Finally, wash in PBT. 0,1% BSA. 2% normal serum for 1 h. 3 lncubale with secondary antibodies in PBT. 0.1% BSA. ~,6 nonnal serum for 211 at ix)ore temperature. Secondary antibodies are conjugated with fllodamine or fluorescein. Dilutions depend on tile secondary antilmdy used, e.g. mcondary antiixfly a~,linst rabbg [gG(H+L) ¢oniu.qatod widl dloc'lanline (Biosys. Bi22flT), 1 : 50, secondary aNtil'~My against muusv 1F,,G(H+L) conjugated with H I E flnsgtut Pa.steur Ptod~actlon), 1 : 200. To reduce background, secondary antilxxlles can Ix' prc-abscdxxl on larval heads dissected, fixed and pemleabilized m'~in tile experiment, Pre-absorh by diluting tile ,~coodary ;mtilxxty 1:8 ha t h r ~ volumes of FBT. 0.1% I~;A, 2% normal senlnl against one volmne o[ fixed ti,~sues, for 2 h :it lX~olnlemperatulx', ,t Wash ixvice in I)BT and e.vice in FBS over a 1 h period. If DNA staining is desired, incubate fls,sues wifl'~the appropriate rtnlgent I~fotc tile PIK";washes. We u.~od 0.5 nlg rul-I dlix)momycin (Sigma C-2659) in PBT for I h at 37°C, which can Ix: detecttxi by confocal imag ng. For convent onal fluores¢cmce i*l cro.scopy, use 0.5-0.1 illgm1-1 DAI , I%PBT for 30 Ill n to 1 h at room ienlpemture. Then, ;~msh P,vice in PBT and twi..'e in PBS o'¢er a I It period. 5 Mount tilt' preparation in Citifluor (Citifluor Lid, London). Discs must he well Ilattencxt to allow obsela,ation. During tile whole process, discs are manlpulmed in glas~s cupels. V(lhnnt.~ art 400 ILl lot washes and 200 ILl for antilx,xly incubations for 10-15 larval heads. Gentle agimtinn is used during tile difl~ercnt int'ubagon and rinse steps. After addition ¢ff fluorescent antilx',dies, incuhatinns are made in tile dark add manipulation can 1~2carried out under a dim grt',2n light, ACKNO~t:U:I)GEMEN'fS We we'.lid like to thank William %~ritfield f¢lr proGding us with Ihe flb188 amilxvdy. This work was supporled by tile Cemre National de la Redlerche Scientiflque tUMR 9922) aod by tile Unive~,~itiesIL aml M, Curie aml D, Did¢~at. REFEBERCI:~ I Stoahl, G. and Basler, K. (1993) Cell7!, 527-540 2 Gonzal~z. C. and Glover, D.M (19931 in T/)e Cell Cyde, A PracticafApp)~lcb (Fmntes, P. and Brtmks, If,, ed.s), pp. 143-175, IRL Press 3 Theurkauf, W,E, t 1992) Dt,v. BioL 154, 20~-2t7 4 Whltfleld, W,G.F, el aL (1988).L c't.I/Sct 89. 467-.180
Cotm*lmtt, d I~l>A,tp~flsAitdif~9"l faltdilvert@cer~u.<~ie~,~.), Altti)t FA,IlvCtold Ma~lfllv Sfl)tottt.liR, D i'uamique du G6nome el Etxdutio., htstitut .[aceplt,$ Moilod, tl~iit~9~it6 Dt.tlL~ Diderot, 2 place Jtt.~ietl, 75005 Parle. arid ~G')olll~' de qt;tldtiqlte Celhdaim et Moh;culaim lIRA CNR$ I L~5 '13iologieMok~ctdaJre el Ck,lhdaO~, d . lX~t~'loplx'me~it" U)lfl%.l~llt~Pfe~. d Marie Clirlt'. 7 ql~ai,Qdlll Bvmatr~ 75005Paris, Fro)ice,
R a p i d PCR-medlated b i d i r e c t i o n a l d e l e t i o n s
-
-
-
51nc~ tile introductkm of PCR, great eltbrt has I~en put into ino~asing the size of its products t. Every new PCR plxRJu~.l must be identified, and ~quencing iX#lll:linstile m a t impotlant nleglod of chamctelizatiun. After t )verconling initial dilllculti~, direct sequencing of FCg ploducLs was eslablisJled as a method of dloi¢,2, Pdther than s~quun¢ing of CIollc~i PCR produc'ts, Inainly ]~cau~¢ tri" thv wcll.
TIG NOVI-~MBER1996 Vt)L. 12 No. 11
453