protocol_294.ann

T1	Action 65 69	Grow
E1	Action:T1 Acts-on:T21 Site:T22
T2	Action 131 134	add
E2	Action:T2 Acts-on:T27 Site:T28
T3	Action 206 214	incubate
E3	Action:T3 
T4	Action 299 304	Rinse
E4	Action:T4 Acts-on:T32 Using:T35
T5	Action 330 333	Fix
E5	Action:T5 Acts-on:T36 Using:T37 Using2:T40
T6	Action 405 410	Rinse
E6	Action:T6 Count:T43 Using:T45
T7	Action 450 455	Block
E7	Action:T7 Acts-on:T47 Using:T49
T8	Action 510 516	Dilute
E8	Action:T8 Acts-on:T52 Using:T56
T9	Action 622 625	add
E9	Action:T9 Site:T62
T10	Action 645 653	incubate
E10	Action:T10 
T11	Action 705 710	Rinse
E11	Action:T11 Count:T64 Using:T66
T12	Action 839 846	Prepare
E12	Action:T12 Creates:T68 Using:T70 Using2:T73
T13	Action 973 976	add
E13	Action:T13 Site:T72 Acts-on:T68
T14	Action 1024 1032	Incubate
E14	Action:T14 Acts-on:T74 Site:T77
T15	Action 1089 1094	Rinse
E15	Action:T15 Count:T78 Site:T80
T16	Action 1134 1143	Coverslip
E16	Action:T16 Using:T82
T17	Action 1176 1180	Seal
E17	Action:T17 Acts-on:T83 Site:T85
T18	Action 1224 1240	prevent movement
E18	Action:T18 Using:E17 Acts-on:T86
T19	Action 1428 1434	stored
E19	Action:T19 Acts-on:T89 Creates:T87
T20	Modifier 70 78	cultured
T21	Reagent 79 84	cells
T22	Location 88 99	cover slips
T23	Location 103 117	chamber slides
T24	Time 118 127	overnight
T25	Modifier 135 146	appropriate
T26	Amount 147 153	amount
T27	Reagent 157 162	cells
T28	Location 166 201	poly‐L‐lysine coated chamber slides
T29	Modifier 215 223	at least
T30	Time 224 234	30 minutes
T31	Temperature 238 242	37°C
R1	Mod-Link Arg1:T21 Arg2:T20	
R2	Or Arg1:T22 Arg2:T23	
R3	Setting Arg1:E1 Arg2:T24	
R4	Measure Arg1:T27 Arg2:T26	
R5	Mod-Link Arg1:T27 Arg2:T25	
R6	Mod-Link Arg1:T30 Arg2:T29	
R7	Setting Arg1:E3 Arg2:T30	
R8	Setting Arg1:E3 Arg2:T31	
T32	Reagent 305 310	cells
T33	Modifier 311 318	briefly
T34	Concentration 322 324	1X
T35	Reagent 325 328	PBS
R9	Measure Arg1:T35 Arg2:T34	
R10	Mod-Link Arg1:E4 Arg2:T33	
T36	Reagent 334 339	cells
T37	Method 343 353	incubation
T38	Modifier 359 363	cold
T39	Concentration 364 368	100%
T40	Reagent 369 377	methanol
T41	Time 382 394	5‐15 minutes
T42	Temperature 398 403	‐20°C
R11	Measure Arg1:T40 Arg2:T39	
R12	Mod-Link Arg1:T40 Arg2:T38	
R13	Setting Arg1:E5 Arg2:T41	
R14	Setting Arg1:E5 Arg2:T42	
T43	Numerical 411 416	three
T44	Concentration 426 428	1X
T45	Reagent 429 432	PBS
T46	Time 434 443	5 minutes
R15	Measure Arg1:T45 Arg2:T44	
R16	Setting Arg1:E6 Arg2:T46	
T47	Reagent 456 463	samples
T48	Concentration 467 469	5%
T49	Reagent 470 477	FBS/PBS
T50	Time 482 488	1 hour
T51	Temperature 492 508	room temperature
R17	Measure Arg1:T49 Arg2:T48	
R18	Setting Arg1:E7 Arg2:T50	
R19	Setting Arg1:E7 Arg2:T51	
T52	Reagent 521 537	primary antibody
T53	Modifier 545 556	recommended
T54	Concentration 557 579	concentration/dilution
R20	Mod-Link Arg1:T54 Arg2:T53	
R21	Setting Arg1:E8 Arg2:T54	
T55	Concentration 583 585	5%
T56	Reagent 586 593	FBS/PBS
R22	Measure Arg1:T56 Arg2:T55	
T57	Amount 626 631	200μl
T58	Time 654 663	2‐3 hours
T59	Temperature 667 683	room temperature
T60	Time 687 696	overnight
T61	Temperature 700 703	4°C
R23	Setting Arg1:E10 Arg2:T58	
R24	Setting Arg1:E10 Arg2:T59	
R25	Or Arg1:T58 Arg2:T60	
R26	Or Arg1:T59 Arg2:T61	
T62	Location 636 640	well
R27	Setting Arg1:E9 Arg2:T57	
T63	Location 599 620	8 well chamber slides
R28	Meronym Arg1:T62 Arg2:T63	
T64	Numerical 711 716	three
T65	Concentration 726 728	1X
T66	Reagent 729 732	PBS
T67	Time 734 743	5 minutes
R29	Measure Arg1:T66 Arg2:T65	
R30	Setting Arg1:E11 Arg2:T67	
T68	Reagent 847 888	fluorophore‐conjugated secondary antibody
T69	Concentration 892 894	5%
T70	Reagent 895 902	FBS/PBS
T71	Amount 977 982	200μl
T72	Location 987 991	well
R31	Measure Arg1:T70 Arg2:T69	
T73	Method 916 968	therecommended manufacturer specification data sheet
R32	Setting Arg1:E13 Arg2:T71	
T74	Reagent 1037 1044	samples
T75	Time 1049 1055	1 hour
T76	Temperature 1059 1075	room temperature
T77	Location 1083 1087	dark
R33	Setting Arg1:E14 Arg2:T75	
R34	Setting Arg1:E14 Arg2:T76	
T78	Numerical 1095 1100	three
T79	Concentration 1110 1112	1X
T80	Reagent 1113 1116	PBS
T81	Time 1118 1127	5 minutes
R35	Measure Arg1:T80 Arg2:T79	
R36	Setting Arg1:E15 Arg2:T81	
T82	Seal 1149 1174	anti‐fade mounting medium
T83	Location 1185 1190	edges
T84	Seal 1198 1207	coverslip
T85	Location 1215 1220	slide
T86	Seal 1248 1257	coverslip
R37	Meronym Arg1:T83 Arg2:T84	
T87	Reagent 1365 1412	Chamber slides and coverslips Fixation solution
T88	Concentration 1414 1418	100%
T89	Reagent 1419 1427	methanol
T90	Temperature 1438 1443	‐20°C
T91	Modifier 1448 1456	at least
T92	Time 1457 1466	two hours
R38	Mod-Link Arg1:T92 Arg2:T91	
R39	Setting Arg1:E19 Arg2:T90	
R40	Setting Arg1:E19 Arg2:T92	
R41	Measure Arg1:T89 Arg2:T88