Unit 4.3 Immunofluorescence Staining

81,7

,PPXQRIOXRUHVFHQFH 6WDLQLQJ

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￥RQ DQG RII PHPEUDQHV LQWR DQG RXW RI WKH QXFOHXV DQG WKURXJK PHPEUDQH WUDIILF SDWKZD\V

%$6,&

35272&2/

,00812)/825(6&(1&( /$%(/,1* 2) &8/785(' &(//6

7KH IROORZLQJ LV D EDVLF |JHQHULF§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¤ODEHOHG JRDW DQWL¤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

0DWHULDOV

&HOOV RI LQWHUHVW JURZLQJ LQ WLVVXH FXOWXUH IRUPDOGHK\GH VHH UHFLSH

3KRVSKDWH EXIIHUHG VDOLQH 3%6 VHH UHFLSH S+

3%6 )%6 3%6 S+ FRQWDLQLQJ IHWDO ERYLQH VHUXP )%6

Z Y VDSRQLQ LQ 3%6 )%6 SUHSDUH IUHVK IURP Z Y VDSRQLQ VWRFN VROXWLRQ $33(1',; $ VWRUH VWRFN XS WR PRQWKV DW °& RU LQ DOLTXRWV XS WR WR \HDUV DW ? °& 3ULPDU\ DQWLERG\

&RQWUROV SUHLPPXQH VHUXP LI XVLQJ UDEELW SRO\FORQDO DQWLERG\ RU DQWLJHQ DGGHG LQ H[FHVV WR SULPDU\ DQWLERG\

6HFRQGDU\ DQWLERGLHV DJDLQVW ,J RI VSHFLHV IURP ZKLFK SULPDU\ DQWLERG\ ZDV REWDLQHG FRXSOHG WR IOXRURSKRUH H J 5,7& UKRGDPLQH LVRWKLRF\DQDWH ),7& IOXRUHVFHQFH LVRWKLRF\DQDWH &\ RU 7H[DV 5HG 0RXQWLQJ PHGLXP VHH UHFLSH

FP GLDPHWHU WLVVXH FXOWXUH GLVKHV

PP QR URXQG JODVV FRYHUVOLSV VWHULOL]HG E\ DXWRFODYLQJ RU VRDNLQJ LQ HWKDQRO

ZHOO WLVVXH FXOWXUH SODWHV PP SHWUL GLVKHV

&RQWULEXWHG E\ -XOLH * 'RQDOGVRQ

&XUUHQW 3URWRFROV LQ &HOO %LRORJ\ &RS\ULJKW E\ -RKQ :LOH\ 6RQV ,QF

0LFURVFRS\

:DWFKPDNHU?V IRUFHSV 0LFURVFRSH VOLGHV 1DLO SROLVK

)OXRUHVFHQFH PLFURVFRSH ZLWK × RLO LPPHUVLRQ OHQV

$GGLWLRQDO UHDJHQWV DQG HTXLSPHQW IRU WU\SVLQL]DWLRQ RI FHOOV 81,7

127( $OO VROXWLRQV DQG HTXLSPHQW FRPLQJ LQWR FRQWDFW ZLWK OLYH FHOOV PXVW EH VWHULOH DQG DVHSWLF WHFKQLTXH VKRXOG EH XVHG DFFRUGLQJO\

127( $OO FXOWXUH LQFXEDWLRQV VKRXOG EH SHUIRUPHG LQ D KXPLGLILHG °& &2 LQFXEDWRU XQOHVV RWKHUZLVH VSHFLILHG 6RPH PHGLD H J '0(0 PD\ UHTXLUH DOWHUHG OHYHOV RI &2 WR PDLQWDLQ S+

)RU DGKHUHQW FHOOV WR GD\V SULRU WR H[SHULPHQW WU\SVLQL]H FHOOV DQG VHHG RQWR FP FXOWXUH GLVKHV HDFK FRQWDLQLQJ WR VWHULOL]HG FRYHUVOLSV VR WKDW RQ GD\ RI H[SHULPHQW FHOOV DUH WR FRQIOXHQW

1RQDGKHUHQW FHOOV FDQ EH FRD[HG LQWR DGKHULQJ WR WKH FRYHUVOLSV E\ SUHFRDWLQJ WKH FRYHUVOLS ZLWK SRO\ / O\VLQH VHH UHFLSH $SSO\ WR μO RI VXVSHQGHG FHOOV WR HDFK FRYHUVOLS OHW VLW PLQ WKHQ SURFHHG ZLWK IL[DWLRQ VWHS

$OWHUQDWLYHO\ FHOOV FDQ EH DWWDFKHG WR FRYHUVOLSV XVLQJ D F\WRFHQWULIXJH E\ IROORZLQJ WKH PDQXIDFWXUHU?V LQVWUXFWLRQV

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

127( 7KURXJKRXW WKH SURFHGXUH GR QRW OHW FHOOV GU\ RXW

,Q PO PLFURFHQWULIXJH WXEHV GLOXWH SULPDU\ DQWLERGLHV LQ VDSRQLQ 3%6 )%6

7\SLFDOO\ DIILQLW\ SXULILHG DQWLERGLHV DUH GLOXWHG LQ WKH UDQJH RI WR μJ PO DQG UDEELW DQWLVHUD DUH GLOXWHG EHWZHHQ DQG ,I XVLQJ D FRPPHUFLDO DQWLERG\ IROORZ VXJJHVWHG GLOXWLRQV IURP PDQXIDFWXUHU

'XULQJ LQLWLDO FKDUDFWHUL]DWLRQ LW LV ZLVH WR WU\ D UDQJH RI GLOXWLRQV RI DQWLERG\

3UHSDUH FRQWUROV FRQWDLQLQJ RQO\ VDSRQLQ 3%6 )%6 RU LI DYDLODEOH FRQWDLQLQJ SUHLPPXQH DQWLVHUXP LI UDEELW SRO\FORQDO DQWLERG\ LV EHLQJ XVHG RU VSHFLILF SULPDU\ DQWLERG\ ZLWK WKH DQWLJHQ DGGHG LQ H[FHVV

&RQWUROV DUH RIWHQ WKH PRVW LPSRUWDQW SDUW RI DQ LPPXQRIOXRUHVFHQFH H[SHULPHQW

0LFURFHQWULIXJH DQWLERG\ GLOXWLRQV DQG FRQWURO VROXWLRQV PLQ DW PD[LPXP VSHHG URRP WHPSHUDWXUH WR EULQJ GRZQ DJJUHJDWHV LQ SHOOHW

3LSHW WKH DQWLERG\ VROXWLRQ IURP DERYH WKH DJJUHJDWH SHOOHW

3ODFH D × ¤FP SLHFH RI 3DUDILOP LQ WKH ERWWRP RI D PP SHWUL GLVK ,Q D JULG SDWWHUQ WKDW UHSOLFDWHV WKH ZHOOV XVHG WR LQFXEDWH WKH FRYHUVOLSV ODEHO WKH DSSUR SULDWH SODFH RQ WKH 3DUDILOP IRU HDFK FRYHUVOLS ZLWK D PDUNHU

&XUUHQW 3URWRFROV LQ &HOO %LRORJ\

,PPXQR IOXRUHVFHQFH

6WDLQLQJ

$SSO\ D μO GURS RI DSSURSULDWH SULPDU\ DQWLERG\ VROXWLRQ WR HDFK QXPEHUHG VHFWLRQ &DUHIXOO\ UHPRYH HDFK FRYHUVOLS IURP WKH ZHOO SODWH ZLWK ZDWFKPDNHU?V IRUFHSV EORW WKH H[FHVV IOXLG E\ WRXFKLQJ WKH HGJH WR D .LPZLSH WKHQ LQYHUW WKH FRYHUVOLS RYHU WKH DSSURSULDWH μO GURS PDNLQJ VXUH WKDW WKH VLGH ZLWK WKH FHOOV LV GRZQ 3ODFH WKH WRS RQ WKH SHWUL GLVK DQG LQFXEDWH KU DW URRP WHPSHUDWXUH

6RPHWLPHV SURSHU ODEHOLQJ ZLOO UHTXLUH D ORQJHU LQFXEDWLRQ WLPH RU WKH SHWUL GLVK ZLOO EH LQFXEDWHG ! KU IRU FRQYHQLHQFH )RU ORQJHU LQFXEDWLRQV DGG VRPH ZHWWHG .LPZLSHV WR WKH GLVK WR PDLQWDLQ D KXPLG DWPRVSKHUH 7KH LQFXEDWLRQ FDQ EH H[WHQGHG RYHUQLJKW DW °& LI QHFHVVDU\

127( ,W LV LPSRUWDQW WR DOZD\V EH DZDUH RI ZKLFK VLGH RI WKH FRYHUVOLS WKH FHOOV DUH RQ &HOOV VKRXOG EH IDFLQJ XS ZKHQ LQ WKH ZHOO SODWH EXW IDFLQJ GRZQ ZKHQ SODFHG RQ WKH DQWLERG\ 3LFNLQJ XS FRYHUVOLSV ZLWK WKH IRUFHSV LV DZNZDUG DW ILUVW EXW EHFRPHV HDVLHU ZLWK SUDFWLFH

&DUHIXOO\ SLFN XS HDFK LQYHUWHG FRYHUVOLS DQG IOLS LW RYHU VR WKDW LW LV FHOO VLGH XS WKHQ SODFH LQ D ZHOO RI D ZHOO SODWH :DVK HDFK FRYHUVOLS WKUHH WLPHV WR UHPRYH XQERXQG DQWLERG\ HDFK WLPH E\ DGGLQJ PO 3%6 )%6 OHWWLQJ VWDQG PLQ WKHQ DVSLUDWLQJ WKH VROXWLRQ

'LOXWH IOXRURSKRUH FRQMXJDWHG VHFRQGDU\ DQWLERGLHV LQ VDSRQLQ 3%6 )%6 0L[ WKHQ PLFURFHQWULIXJH DV LQ VWHS WR UHPRYH DJJUHJDWHV

7\SLFDOO\ FRPPHUFLDO SUHSDUDWLRQV DUH GLOXWHG EHWZHHQ DQG

3UHSDUH DQ LQFXEDWLRQ FKDPEHU DV LQ VWHS $SSO\ μO RI DSSURSULDWH VHFRQGDU\ DQWLERG\ VROXWLRQ WR HDFK QXPEHUHG VHFWLRQ DQG LQYHUW FRYHUVOLS RYHU GURS DV LQ VWHS &RYHU SHWUL GLVK DQG SURWHFW IURP OLJKW ZLWK DOXPLQXP IRLO RU SODFH FKDPEHU LQ GUDZHU ,QFXEDWH KU DW URRP WHPSHUDWXUH

:DVK FRYHUVOLSV DV LQ VWHS $IWHU UHPRYDO RI ODVW 3%6 )%6 ZDVK DGG PO 3%6 /DEHO VOLGHV DQG SODFH GURS RI PRXQWLQJ PHGLXP RQWR VOLGH 3LFN XS FRYHUVOLS IURP ZHOO JHQWO\ EORW RII H[FHVV 3%6 E\ WRXFKLQJ WKH HGJH WR D .LPZLSH WKHQ LQYHUW FRYHUVOLS FHOO VLGH GRZQ RQWR GURS *HQWO\ EORW PRXQWHG FRYHUVOLS ZLWK SDSHU WRZHO WKHQ VHDO HGJH RI FRYHUVOLS RQWR VOLGH E\ SDLQWLQJ WKH HGJH ZLWK D ULP RI QDLO SROLVK /HW GU\

7KH IL[HG PRXQWHG DQG QDLO SROLVK¤VHDOHG FRYHUVOLSV FDQ EH VWRUHG LQ WKH GDUN IRU PRQWKV WR \HDU DW °&

9LHZ VSHFLPHQ RQ IOXRUHVFHQFH PLFURVFRSH XVLQJ DQ × RLO LPPHUVLRQ REMHFWLYH 5($*(176 $1' 62/87,216

8VH GHLRQL]HG RU GLVWLOOHG ZDWHU LQ DOO UHFLSHV DQG SURWRFRO VWHSV )RU FRPPRQ VWRFN VROXWLRQV VHH

$33(1',; $ IRU VXSSOLHUV VHH 6833/,(56 $33(1',;

)RUPDOGHK\GH

,Q D FKHPLFDO IXPH KRRG GLOXWH PO UHDJHQW JUDGH IRUPDOGHK\GH LQWR PO 3%6 VHH UHFLSH S+

$V UHDJHQW JUDGH IRUPDOGHK\GH FRQWDLQV PHWKDQRO DV DQ DOWHUQDWLYH RU LI QHFHVVDU\ PDNH XS IRUPDOGHK\GH IURP SDUDIRUPDOGHK\GH E\ GLVVROYLQJ J SDUDIRUPDOGHK\GH SRZGHU LQ PO + 2 WKDW KDV EHHQ KHDWHG WR °& WKHQ GLOXWLQJ ZLWK × 3%6 S+ ,W PLJKW EH GHVLUDEOH WR WU\ ERWK SURFHGXUHV IRU SUHSDULQJ WKH IRUPDOGHK\GH VROXWLRQ DQG VHH ZKLFK JLYHV EHWWHU UHVXOWV

0RXQWLQJ PHGLXP

8VH )OXRUPRXQW * 6RXWKHUQ %LRWHFKQRORJ\ RU SUHSDUH Z Y JO\FHURO DQG Z Y S SKHQ\OHQHGLDPLQH LQ 3%6 VHH UHFLSH S+

&XUUHQW 3URWRFROV LQ &HOO %LRORJ\

0LFURVFRS\

3KRVSKDWH EXIIHUHG VDOLQH 3%6

J .+ 32 J 1D&O

J 1D +32 ? + 2+ 2 WR OLWHU

$GMXVW WR GHVLUHG S+ ZLWK 0 1D2+ RU 0 +&O 6WRUH LQGHILQLWHO\ DW URRP WHPSHUDWXUH

3RO\ / O\VLQH FRDWHG FRYHUVOLSV

$SSO\ μO RI PJ PO SRO\ / O\VLQH WR HDFK VWHULOL]HG QR FRYHUVOLS LQ D KRRG DQG DOORZ WR VWDQG ～ PLQ &DUHIXOO\ ULQVH FRYHUVOLSV WKUHH WLPHV ZLWK ZDWHU WKHQ DOORZ WR DLU GU\ &200(17$5<%DFNJURXQG ,QIRUPDWLRQ

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

&ULWLFDO 3DUDPHWHUV

7R HQVXUH VXFFHVV ZLWK LPPXQRIOXRUHV FHQFH WKUHH SDUDPHWHUV DUH FULWLFDO￥IL[DWLRQ SHUPHDELOL]DWLRQ DQG GHWHUPLQDWLRQ RI WKH VSH FLILFLW\ RI ODEHOLQJ 7KH IL[DWLRQ DQG SHUPHDEL

OL]DWLRQ FRQGLWLRQV PXVW EH DVVHVVHG LQGLYLGX DOO\ IRU HDFK DQWLERG\ DQG HDFK FHOO W\SH LQYHVW LJDWHG 5HIHU WR *ULIILWKV IRU DQ H[FHOOHQW GLVFXVVLRQ RQ IL[DWLRQ DQG LVVXHV UHODWHG WR VSHFLILFLW\ RI DQWLERG\ ODEHOLQJ

'LIIHUHQW IL[DWLYHV PLJKW EH LQYHVWLJDWHG WR RSWLPL]H SUHVHUYDWLRQ RI WKH DQWLJHQ LWV GLVWUL EXWLRQ DQG WKH PRUSKRORJ\ RI RWKHU FHOOXODU FRQVWLWXHQWV )RU H[DPSOH VRPH HSLWRSHV DUH ORVW XSRQ DOGHK\GH IL[DWLRQ EXW SUHVHUYHG ZLWK DO FRKRO IL[DWLRQ DQG YLFH YHUVD $OWHUQDWLYH IL[D WLYHV WR WU\ LQFOXGH PHWKDQRO DW ? °& PLQ H[SRVXUH RU IRUPDOGHK\GH IL[DWLRQ IROORZHG E\ D EULHI PLQ H[SRVXUH WR PHWKDQRO DW °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

7KH SULPDU\ DQWLERG\ VKRXOG EH SXULILHG WR WKH H[WHQW WKDW LW UHFRJQL]HV RQO\ WKH SURWHLQ RI LQWHUHVW RQ LPPXQREORWV $OWKRXJK DIILQLW\ SX

&XUUHQW 3URWRFROV LQ &HOO %LRORJ\

,PPXQR IOXRUHVFHQFH

6WDLQLQJ

ULILFDWLRQ RIWHQ UHVROYHV WKLV EH DZDUH WKDW |DIILQLW\ SXULILHG§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|VSHFLILF ODEHOLQJ§ ZLWK WKH DQWLERG\ 7R DVVHVV WKLV FRQWURO VWDLQHG VOLGHV LQFXEDWHG ZLWK VHFRQGDU\ DQWLERG\ RQO\ RU ZLWK SUHLPPXQH VHUXP VKRXOG ILUVW EH YLHZHG WR GHWHUPLQH ZKDW FRQVWLWXWHV |QRQVSHFLILF VWDLQLQJ §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

REVHUYHG LV GXH WR WKH SUHVHQFH RI WKH SURWHLQ RI

)LJXUH ([DPSOHV RI LPPXQRIOXRUHVFHQFH ODEHOLQJ RI IRUPDOGHK\GH IL[HG FHOOV $ DQG % 'RXEOH ODEHOLQJ RI D QRUPDO UDW NLGQH\ FHOO ZLWK D PRXVH PRQRFORQDO DQWLERG\ WR $ WKH β &23 FRPSRQHQW RI FRDWRPHU DQG % D UDEELW SRO\FORQDO DQWLERG\ WR PDQQRVLGDVH ,, & 'LVWULEXWLRQ RI YLQFXOLQ LQ D IRUPDOGHK\GH IL[HG QRUPDO UDW NLGQH\ FHOO XVLQJ D PRXVH PRQRFORQDO DQWLERG\ ' 'LVWULEXWLRQ RI WUDQVIHUULQ UHFHSWRU LQ IRUPDOGHK\GH IL[HG +H/D FHOOV XVLQJ D PRXVH PRQRFORQDO DQWLERG\ %DU LV HTXDO WR μP

&XUUHQW 3URWRFROV LQ &HOO %LRORJ\

0LFURVFRS\

LQWHUHVW ,I D UDEELW DQWLVHUXP LV XVHG SUHLP PXQH VHUXP VKRXOG \LHOG D SDWWHUQ RI VWDLQLQJ VLPLODU WR WKDW RI WKH VHFRQGDU\ DQWLERG\ DORQH ,I WKH LPPXQL]LQJ DQWLJHQ LV DYDLODEOH LW FDQ EH DGGHG WR WKH DQWLERG\ GLOXWLRQ WR FRPSHWH IRU WKH VSHFLILF VWDLQLQJ DQG LW VKRXOG \LHOG |EDFN JURXQG§ OHYHOV RI IOXRUHVFHQFH /DVWO\ VXSSRUW IRU VSHFLILF ODEHOLQJ FDQ EH FRQILUPHG E\ ODFN RI VWDLQLQJ LQ D FHOO W\SH WKDW GRHV QRW H[SUHVV WKH SURWHLQ RI LQWHUHVW DQG LQFUHDVHG ODEHOLQJ LQ FHOOV RYHUH[SUHVVLQJ WKH SURWHLQ E\ WUDQVLHQW WUDQVIHFWLRQ )LQDOO\ WKH XVH RI GLIIHUHQW DQWL ERGLHV WR WKH SURWHLQ LI DYDLODEOH FDQ LQGH SHQGHQWO\ FRQILUP WKH ORFDOL]DWLRQ SDWWHUQ

7URXEOHVKRRWLQJ

,I EDFNJURXQG VWDLQLQJ ZLWK VHFRQGDU\ DQWL ERGLHV LV WRR KLJK WKHUH DUH VHYHUDO SRVVLEOH UHPHGLHV 7KH VHFRQGDU\ DQWLERGLHV PD\ EH GLOXWHG IXUWKHU RU GLIIHUHQW VHFRQGDU\ DQWLERG LHV GLIIHUHQW KRVWV RU GLIIHUHQW IOXRURSKRUHV PD\ EH WULHG $OVR LW LV SRVVLEOH WR WU\ RWKHU |VRUELQJ§ UHDJHQWV 3%6 )%6 LV JHQHUDOO\ D JRRG VRUELQJ UHDJHQW WKDW HIIHFWLYHO\ EORFNV RXW QRQVSHFLILF VLWHV EXW RWKHU DJHQWV FDQ EH XVHG￥H J Z Y %6$ LPPXQRJOREXOLQ IUHH RU JHODWLQ

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¤PRXVH ,J* DQG WKHQ LQ ),7& FRQMXJDWHG GRQ NH\ DQWL¤JRDW ,J* &DXWLRQ VKRXOG EH REVHUYHG WKDW VHFRQGDU\ DQWLERGLHV GR QRW DOWHU WKH SDW WHUQ RI VLQJOH DQWLERG\ ODEHOLQJ

$QWLFLSDWHG 5HVXOWV

2QFH FRQGLWLRQV IRU VSHFLILF ORFDOL]DWLRQ KDYH EHHQ RSWLPL]HG WKH GLVWULEXWLRQ RI WKH SURWHLQ LQ D YDULHW\ RI FHOOV XQGHU D YDULHW\ RI FRQGLWLRQV FDQ EH REVHUYHG VHH )LJ 'RXEOH ODEHO LQJ RI WZR DQWLJHQV DOORZV WKH GLVWULEXWLRQ RI RQH SURWHLQ WR EH FRPSDUHG ZLWK WKDW RI DQRWKHU SURWHLQ LQ WKH VDPH VDPSOH XVLQJ WZR VHFRQGDU\DQWLERGLHV DWWDFKHG WR IOXRURSKRUHV WKDW FDQ EH

PRQLWRUHG LQ WZR GLIIHUHQW FKDQQHOV E\ IORZ F\WRPHWU\ 7KXV WKH ORFDWLRQ RI D NQRZQ SUR WHLQ VD\ DQ DQWLERG\ WR D *ROJL UHVLGHQW SUR WHLQ FDQ EH XVHG DV D PDUNHU IRU WKH *ROJL FRPSOH[ WR VHH ZKHWKHU WKH DQWLJHQ XQGHU VWXG\FRORFDOL]HV ZLWK LW )LJ SDQHOV $ DQG %

7LPH &RQVLGHUDWLRQV

7KH HQWLUH SURFHGXUH FDQ EH SHUIRUPHG LQ ～ KU )L[HG ZDVKHG FRYHUVOLSV FDQ EH VWRUHG DW °& IRU VHYHUDO GD\V SULRU WR LPPXQROD EHOLQJ ,QFXEDWLRQV ZLWK SULPDU\ DQWLERGLHV FDQ EH H[WHQGHG WR RYHUQLJKW DW °& LI QHFHVVDU\ RU LI LW LV GHVLUDEOH WR LQFUHDVH VWDLQLQJ 'LVFODLPHU

7KLV DUWLFOH ZDV ZULWWHQ E\ -XOLH 'RQDOGVRQ LQ KHU SULYDWH FDSDFLW\ 1R RIILFLDO VXSSRUW RU HQGRUVHPHQW E\ WKH 1+/%, RU 1,+ LV LQWHQGHG DQG QRQH VKRXOG EH LQIHUUHG

/LWHUDWXUH &LWHG

&KDYULHU 3 3DUWRQ 5 * +DXUL + 3 6LPRQV . DQG =HULDO 0 /RFDOL]DWLRQ RI ORZ PROHFX ODU ZHLJKW *73 ELQGLQJ SURWHLQV WR H[RF\WLF DQG HQGRF\WLF FRPSDUWPHQWV &HOO &RRQV $ + 7KH EHJLQQLQJV RI LPPXQRIOXR UHVFHQFH - ,PPXQRO *ULIILWKV * )LQH 6WUXFWXUH ,PPXQRF\WR FKHPLVWU\ 6SULQJHU 9HUODJ +HLGHOEHUJ /LSSLQFRWW 6FKZDUW] - 'RQDOGVRQ - * 6FKZHL]HU $ %HUJHU ( * +DXUL + 3

&RQWULEXWHG E\ -XOLH * 'RQDOGVRQ 1DWLRQDO +HDUW /XQJ DQG %ORRG ,QVWLWXWH 1,+%HWKHVGD 0DU\ODQG

&XUUHQW 3URWRFROV LQ &HOO %LRORJ\

,PPXQR IOXRUHVFHQFH

6WDLQLQJ