1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
Additional File 2: Detailed protocols for fixation and
immunofluorescent labeling of planarians
David J. Forsthoefel, Forrest A. Waters, and Phillip A. Newmark
Contents:
Magnesium relaxation...........................................................2
N-Acetyl cysteine mucus removal.........................................3
HCl mucus removal...............................................................3
Methacarn fixation.................................................................4
Formaldehyde fixation...........................................................4
Reduction..............................................................................5
Hydrogen peroxide bleaching...............................................6
Proteinase K treatment..........................................................7
Antigen retrieval.....................................................................7
Immunolabeling.....................................................................8
Tyramide Signal Amplification (TSA).....................................9
Cryosectioning.....................................................................10
Gelatin/CPS slide coating....................................................12
Rehydration (cryosections)..................................................13
Antigen retrieval (cryosections)...........................................13
Reduction (cryosections).....................................................13
Proteinase K treatment (cryosections).................................14
Immunolabeling (cryosections)............................................15
Sample whole animal optimization.......................................16
Sample whole animal labeling (mAb 3F11)..........................17
Sample cryosection protocol (mAb 3F11)............................18
Optimal protocols for selected mAbs.(whole animals).........19
Optimal protocols for selected mAbs.(cryosections)............20
Working Solutions................................................................21
Reagents/Equipment............................................................22
References...........................................................................23
Forsthoefel DJ, Waters FA, and Newmark PA, 2014
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
61
62
63
64
65
66
67
68
69
70
71
72
73
74
75
76
77
Magnesium relaxation
1. Place 5-10 animals in a small glass vial, or 20-30 in a large vial.
2. Remove all but 0.5ml/1ml planarian salts (small/large vial).
3. While animals are moving, add 1ml/2ml 1M MgCl2 (final conc. = 0.67M).
4. Swirl gently for 15-30 sec maximum. Animals will first contract, then relax and
uncurl.
5. Once animals have uncurled1, fill remainder of vial with 1X PBS and swirl.
Animals will sink and collect in the center of the bottom.
6. Quickly2 but carefully remove all MgCl2/PBS, so that most animals flatten on
the bottom/sides of the vial (use a transfer pipet followed by a P100 pipet for the
last few drops). Proceed immediately to HCl or NAc mucus removal.
Notes:
1
Usually only 80-90% of animals will properly uncurl/flatten, so start with more
than are needed for the experiment.
2
The entire Mg step must be completed within 90-120 sec or animals will begin
to lyse due to (presumably) osmotic shock.
2
Forsthoefel DJ, Waters FA, and Newmark PA, 2014
78
79
80
81
82
83
84
85
86
87
88
89
90
91
92
93
94
95
96
97
98
99
100
101
102
103
104
105
106
107
108
109
110
111
112
113
114
115
116
117
118
119
120
121
Mucus removal with N-Acetyl Cysteine (Adapted from [1])
(skip step 1 if animals have been relaxed in magnesium)
1. Place 5-10 animals in a small glass vial, or 20-30 in a large vial. Remove salts.
2. Add 5 ml/10 ml (small/large vial) 7.5% NAc/PBS. Incubate with gentle rocking
for 7 min.
3. Remove NAc.
4. Add fixative.
Mucus removal with HCl (Adapted from [2])
(skip steps 1-2 if animals have been relaxed in magnesium)
1. Place 5-10 animals in a small glass vial, or 20-30 in a large vial.
2. Incubate animals on ice 30 sec – 2 min, until movement slows considerably.
Quickly remove salts.
3. Add 5 ml/10 ml (small/large vial) ice-cold 2% HCl (diluted into ultra-pure
water).
4. Shake 1 min, incubate on ice 1 min, shake for final minute (3 min total).
5. Quickly remove all HCl. If fixing in formaldehyde, quickly rinse animals in 1X
PBS1 and remove.
6. Add fixative.
Note:
1 This
step prevents interaction between HCl and formaldehyde to form the
carcinogen bis-chloromethyl ether (BCME).
3
Forsthoefel DJ, Waters FA, and Newmark PA, 2014
122
123
124
125
126
127
128
129
130
131
132
133
134
135
136
137
138
139
140
141
142
143
144
145
146
147
148
149
150
151
152
153
154
155
156
157
158
159
160
161
Methacarn fixation
1. Add 5ml/10ml (small/large vial) ice-cold methacarn.
2. Shake moderately by hand for 1 min, or until animals sink.
3. Rock gently for 20 min at 4C.
4. Remove methacarn, add -20C methanol, rock 15-30 min at 4C.
5. Remove methanol, rinse animals 1-2 times in methanol, then proceed with
bleaching or storage at -20C.
Formaldehyde fixation
1. Add 5 ml/10 ml (small/large vial) 4% formaldehyde solution.
2. Rock gently at RT for 20 min.
3. Remove fixative, then rinse 3 times in PBSTx, 5 min each1.
4. Proceed with storage1 or bleaching.
Notes:
1
Animals can be stored for up to a week in PBSTx at 4C, or dehydrated into
methanol and stored indefinitely at -20C in methanol. Storage effects should be
tested for individual antibodies. See Note 1 in “Hydrogen peroxide bleaching”
(page 5) for details on dehydration.
4
Forsthoefel DJ, Waters FA, and Newmark PA, 2014
162
163
164
165
166
167
168
169
170
171
172
173
174
175
176
177
178
179
180
181
Reduction (Adapted from [3])
1. Rinse animals in PBSTx1.
2. Add freshly-made reduction solution, incubate at 37C for 10 min with gentle
inversion every 2-3 minutes.
3. Wash 3 times with PBSTx, 5 min per wash.
4. Proceed with bleaching.
Note:
1
Reduction is conducted after fixation, prior to bleaching. Animals can be
transferred to standard 1.5 ml polypropylene microcentriguge tubes for this step.
5
Forsthoefel DJ, Waters FA, and Newmark PA, 2014
182
183
184
185
186
187
188
189
190
191
192
193
194
195
196
197
198
199
200
201
202
203
204
205
206
207
208
209
210
211
212
213
Hydrogen peroxide bleaching
1. Add 6% H2O2/methanol or 6% H2O2/PBS to animals, invert several times to
mix.1
2. Bleach in a tinfoil-lined container, 10-15 cm under a fluorescent lamp, for 8-24
hr2.
3. Rinse animals 3 times in methanol or PBS, 5 min each.
4. Store animals at -20C (in methanol) or at 4C (in PBS), or proceed with
rehydration (for animals in methanol) and immunolabeling.
Note:
1
Dehydration/rehydration: If necessary, rehydrate animals by washing in 1:1
methanol:PBSTx (5 min), followed by two washes in PBSTx, 5 min each.
Dehydrate animals by washing in methanol:PBSTx (5 min), followed by two
washes in methanol, 5 min each. For example, after methacarn fixation, animals
should first be rehydrated into PBS before bleaching in 6% H2O2/PBS.
Conversely, after FA fixation, dehydrate animals prior to bleaching in 6%
H2O2/methanol.
2
Bleaching time should be optimized for animal size, fixation conditions, and
individual antibodies.
6
Forsthoefel DJ, Waters FA, and Newmark PA, 2014
214
215
216
217
218
219
220
221
222
223
224
225
226
227
228
229
230
231
232
233
234
235
236
237
238
239
240
241
242
243
244
245
246
247
248
249
250
251
252
253
254
255
256
257
258
259
Proteinase K treatment (Adapted from [3])
1. If necessary, rehydrate animals by washing in 1:1 methanol:PBSTx (5 min),
followed by two washes in PBSTx, 5 min each.
2. Add proteinase K solution, incubate with gentle rocking at RT for 10 min1.
3. Remove proteinase K, and rinse animals 2-3 times in PBSTx.
4. Immediately post-fix for 10 min in 4% formaldehyde solution.
5. Rinse animals 3 times in PBSTx, 5 min each.
Note:
1 Optimized
for intact asexuals, 3-5 mm. Proteinase K concentration and/or
treatment time may need to be adjusted for larger or smaller animals, or for
specific antibodies.
Antigen retrieval
1. If necessary, rehydrate animals1 (see Step 1, above, for proteinase K
treatment.)
2. Equilibrate animals 2 times in AR solution at RT, 5 min each.
3. Incubate animals in AR solution in a heat block at 100°C for 10 min.
4. Allow animals to cool gradually to RT.
5. Rinse animals 2 times in PBSTx, 5 min each.
Note:
1 AR
can be performed before or after bleaching. We have not tested AR
extensively on whole planarians. Furthermore, although AR exposes antigens on
cryosections, we have not yet identified antigens that benefit from this treatment
in whole (bleached) planarians.
7
Forsthoefel DJ, Waters FA, and Newmark PA, 2014
260
261
262
263
264
265
266
267
268
269
270
271
272
273
274
275
276
277
278
279
280
281
282
283
284
285
286
287
288
289
290
291
292
293
294
295
296
297
298
299
Immunolabeling
1. Rehydrate animals if needed (1:1 methanol:PBSTx for 5 min, then 2 washes in
PBSTx, 5 min each).
2. Incubate 4-16 hr1 in blocking solution, at RT.
3. Dilute mAb (1:1-1:100, as appropriate)2 in blocking solution3.
4. Incubate animals in mAb solution for 12-16 hr with gentle rocking at 4C.
5. Remove mAb solution, wash animals 6-8 times in PBSTx over >6 hr at RT.
6. Reblock 30-60 min in blocking solution.
7. Mix secondary antibody solution: HRP-conjugated goat anti-mouse IgG+IgM
(H+L) 1:250 (Jackson), plus DAPI at 1 μg/ml in blocking buffer.
8. Incubate animals in secondary antibody solution for 12-16 hr with gentle
rocking at 4°C.
9. Wash animals 6-8 times in PBSTx over >6 hr at RT.
10. Proceed with tyramide signal amplification.
Notes:
1 We
routinely block “overnight” (12-16 hr), which moderately improves labeling
with intestinal antibodies. Non-intestinal antibodies can be used after blocking for
4 hr, but longer is not detrimental.
2
See Supplemental Table 1. Optimal dilution should be determined for each
batch of supernatant.
75-100 μl solution is sufficient for 3-4 small asexuals in one well of a 96 well
plate.
3
8
Forsthoefel DJ, Waters FA, and Newmark PA, 2014
300
301
302
303
304
305
306
307
308
309
310
311
312
313
314
315
316
317
318
319
320
321
322
323
324
325
326
Tyramide Signal Amplification
(Adapted from [3-5])
1. Wash twice in PBSTw, 5 min each.
2. Add tyramide solution to animals (100 μl minimum in 96 well plates).
3. Incubate in dark for 10 minutes1.
4. Remove tyramide solution.
5. Wash 3 times in PBSTx, 10 min each, then overnight at 4C.
6. Rinse in PBSTx, and mount in Vectashield or other medium.
Note:
1
Tyramide-only preincubation and/or longer development times may improve
labeling in animals over 5 mm in length.
9
Forsthoefel DJ, Waters FA, and Newmark PA, 2014
327
328
329
330
331
332
333
334
335
336
337
338
339
340
341
342
343
344
345
346
347
348
349
350
351
352
353
354
355
356
357
358
359
360
361
362
363
364
365
366
367
368
369
370
371
372
Cryosectioning
1. Cryoprotect. In a polypropylene microcentrifuge tube, incubate appropriately
fixed and processed animals in 15% sucrose solution for 10 min at RT. Replace
with 30% sucrose solution, incubate for 10 min at RT or overnight at 4°C1.
2. Mount. Fill the well of a silicone embedding mold (e.g., “Pelco 106” from Ted
Pella) with TBS Tissue Freezing Medium ("mounting medium")2.
3. Transfer 1-4 planarians to the surface of the mounting medium (use a pipet tip
cut with a razor blade). Remove as much excess 30% sucrose as possible.
4. Orient animals appropriately with a tungsten needle or other probe (see
diagram below). Remove excess mounting medium so that surface is flat or
slightly convex.
5. Freeze entire mold on a flat block of dry ice immediately3.
6. Cryosection4. Apply mounting medium to metal chuck. Remove individual
blocks from the silicone mold, orient appropriately, and freeze to the chuck (see
diagram below). Cut sections (typically 20 μm) and apply to gelatin/CPS coated
SuperFrost Plus slides. Air-dry slides for 1 – 3 hr (i.e., as long as it takes to
section).
6. Store slides in a plastic slide box (Ted Pella), sealed in a ziplock bag, at 80°C5.
Notes:
1 For
best results, cryoprotect planarians immediately after fixation or bleaching.
Animals can be stored at 4°C for several weeks, although some antigens might
be more or less sensitive to storage.
2 We
have found that “TFM” curls less than the more widely used OCT,
particularly for small blocks. Animals can be equilibrated in freezing medium prior
to mounting, but this step has little effect on morphology or immunolabeling.
3
Frozen blocks can be stored at -80°C indefinitely for many antibodies.
4
Equipment and procedures vary among institutions. Accordingly, we have
provided only a general outline. For small blocks, we have found that sectioning
at -25°C reduces curling and yields more consistent sections.
5
We have successfully labeled sections after three months of storage at -80°C.
10
Forsthoefel DJ, Waters FA, and Newmark PA, 2014
373
374
375
376
377
378
379
380
381
382
383
384
385
386
Cryosectioning (continued)
Mounting cryosections. (A) Fixed planarians are transferred to a well of a
silicone mold filled with mounting medium, then oriented so that the anterior tip of
the animal aligns with the tapered end of the well/block. (B) Mounting medium is
added, and the block is frozen to the chuck so that the animal is positioned
perpendicularly to the cutting plane (to generate cross sections).
11
Forsthoefel DJ, Waters FA, and Newmark PA, 2014
387
388
389
390
391
392
393
394
395
396
397
398
399
400
401
402
403
404
405
406
407
408
409
410
411
412
Gelatin/CPS slide coating
1. Heat 500 ml of distilled water in a microwave to 40°C.
2. Add 2.5 gm of gelatin; stir to dissolve.
3. Add 250 mg of chromium (III) potassium sulfate (“CPS”); stir to dissolve 1.
4. Mount several boxes of SuperFrost Plus slides in staining racks (e.g., Ted
Pella 21078).
5. Pour gelatin/CPS solution into staining box (e.g. Ted Pella 21078-1).
6. Dip slides for 5-10 sec and dab away excess on paper towels. Air-dry slides
overnight2.
Notes:
1
Remove excess foam from the solution using a glass rod and Kim wipes prior to
coating slides.
2
Performing this step in a hood will minimize dust contamination on slides.
12
Forsthoefel DJ, Waters FA, and Newmark PA, 2014
413
414
415
416
417
418
419
420
421
422
423
424
425
426
427
428
429
430
431
432
433
434
435
436
437
438
439
440
441
442
443
444
445
446
447
448
449
450
451
452
453
454
455
456
457
Cryosection rehydration
1. Remove slides from -80°C, allow to equilibrate to room temperature for 10 min.
2. Rehydrate sections (and dissolve mounting medium) in 1X PBS in glass
staining jars: 3 washes, 5-10 min each.
Antigen retrieval (slides)
1. Rehydrate sections, then equilibrate in AR solution (5 min).
2. Transfer slides to AR solution in plastic, microwave-safe Coplin jars.
3. Microwave. Heat solution just until boiling begins, being very careful not to
superheat or allow AR solution to boil over. (Heating on lower power can also
minimize superheating.) Then heat every minute for 5-10 sec to bring back to just
boiling. Repeat for 10 min.
4. Allow slides to cool to RT gradually (30-45 min).
5. Wash slides 2 times in PBSTx, 5 min each.
Reduction (slides)
1. Rehydrate sections, then equilibrate in PBSTx (5 min).
2. Remove slide from staining jar, and quickly dab away excess PBSTx from
bottom and sides of slide with a Kimwipe.
3. Add 50 μl reduction solution, and cover sections with 22x50 mm glass
coverslips, using fine forceps.
4. Incubate at 37°C for 10 min.
5. Dip slides in PBSTx in staining jars to remove coverslips.
6. Wash 3 times in PBSTx, 5 min each.
13
Forsthoefel DJ, Waters FA, and Newmark PA, 2014
458
459
460
461
462
463
464
465
466
467
468
469
470
471
472
473
474
475
476
477
478
479
480
Proteinase K treatment (slides)
1. Rehydrate sections, then equilibrate in PBSTx (5 min).
2. Remove slide from staining jar, and quickly dab away excess PBSTx from
bottom and sides of slide with a Kimwipe.
3. Add 50 μl Proteinase K solution, and cover sections with 22x50 mm glass
coverslips, using fine forceps.
4. Incubate at RT for 5 min.
5. Dip slides in PBSTx in staining jars to remove coverslips.
6. Rinse 2 times in PBSTx, 15-30 sec each.
7. Post-fix immediately with 50 μl 4% formaldehyde solution, 5 min.
8. Remove coverslips, wash 3 times in PBSTx, 5 min each.
14
Forsthoefel DJ, Waters FA, and Newmark PA, 2014
481
482
483
484
485
486
487
488
489
490
491
492
493
494
495
496
497
498
499
500
501
502
503
504
505
506
507
508
509
510
511
512
513
514
515
516
517
518
519
520
521
522
523
524
Immunolabeling (cryosections)
1. Equilibrate slides in PBSTx (5 min) in a glass staining jar.
2. Incubate slides in blocking solution for 30 min in a plastic staining jar.
3. Dilute mAb into blocking solution, 50 μl per slide.
4. Remove slides from staining jar, and dab away excess blocking solution with a
Kimwipe. Add mAb solution (50 μl), and cover sections with a 22x50 mm cover
glass using fine forceps.
5. Incubate in a humidified staining chamber1 for 2 hr at RT.
6. Gently remove coverslips by dipping in PBSTx. Rinse slides in fresh PBSTx,
15-30 sec, to remove excess antibody.
7. Wash slides 3 times in PBSTx, 10 min each.
8. Dab away excess PBSTx, add secondary antibody solution (HRP-conjugated
goat anti-mouse IgG+IgM, 1:250, plus DAPI at 1 μg/ml), 50 μl per slide, under a
coverslip.
9. Incubate in a humidified staining chamber for 2 hr at RT.
10. Remove coverslips, rinse slides, and wash 3 times in PBSTx, 10 min each,
as above.
11. Wash slides 2 times in PBSTw, 5 min each.
12. Dab away excess PBSTx, add 50 μl tyramide solution and coverslip. Incubate
10 min at RT.
13. Remove coverslips, rinse slides, and wash 3 times in PBSTx, 10 min each.
14. Cover sections with Vectashield (~22 μl) and 22x50 mm cover glass.
Note:
1 For
example, a plastic “Tuff Tainer” with a moistened paper towel.
15
Forsthoefel DJ, Waters FA, and Newmark PA, 2014
525
526
527
528
529
530
531
532
533
534
535
536
537
538
539
540
541
542
543
544
545
546
547
548
549
550
551
552
553
554
555
556
557
558
Sample optimization protocol (whole animals)
The following is a sample protocol that evaluates 6 combinations of mucus
removal, fixation, and bleaching diluent for two different mAb supernatant
concentrations. Eight small (3-5 mm) asexual planarians are fixed per
combination, then divided into two wells of a 96 well plate for immunofluorescent
labeling. 48 animals total are used.
1. Process 6 sets of samples1, 8 animals per vial:
Abbreviation
HM-P
HF-P
NF-P
HM-M
HF-M
NF-M
Mucus Removal
2% HCl (3 min)
2% HCl (3 min)
7.5% NAc (7 min)
2% HCl (3 min)
2% HCl (3 min)
7.5% NAc (7 min)
Fixation
Methacarn (20 min)
4% Formaldehyede (20 min)
4% Formaldehyede (20 min)
Methacarn (20 min)
4% Formaldehyede (20 min)
4% Formaldehyede (20 min)
Bleach
6% H2O2/PBS (12 hr)
6% H2O2/PBS (12 hr)
6% H2O2/PBS (12 hr)
6% H2O2/MeOH (12 hr)
6% H2O2/MeOH (12 hr)
6% H2O2/MeOH (12 hr)
2. Wash, rehydrate (MeOH-bleached samples), and aliquot each set of animals
to two wells of a 96 well plate (4 animals per well), 12 wells total:
4. Block 4-16 hr.
5. Prepare mAb solutions:
(a) 1:2, 6 wells, 100 μl per well = 300 μl supernatant + 300 μl blocking solution
(b) 1:10, 6 wells, 100 μl per well = 60 μl supernatant + 560 μl blocking solution
6. Remove block, add mAb. Incubate overnight, 4°C.
7. Remove mAb, rinse wells with PBSTx, then wash 6 times in PBSTx, 1 hr each.
8. Re-block 1 hr.
9. Incubate in goat anti-mouse HRP, 1:250, plus DAPI at 1 μg/ml (100 μl/well).
10. Rinse all wells, then wash 6 times in PBSTx, 1 hr each.
11. Wash 2 times in PBSTw.
12. TSA, 10 min.
13. PBSTx washes.
14. Mount in Vectashield.
Note:
1
For simplicity, abbreviated protocols are shown that do not include washes, etc.
16
Forsthoefel DJ, Waters FA, and Newmark PA, 2014
559
560
561
562
563
564
565
566
567
568
569
570
571
572
573
574
575
576
577
578
579
580
581
582
583
584
585
586
587
588
589
590
591
592
593
Sample Protocol, mAb 3F11 (whole animals)
Processing:
1. Relax 3-5 mm asexual planarians in MgCl2.
2. Remove mucus in 2% HCl (3 min), then rinse quickly in PBS.
3. Fix in 4% formaldehyde/PBSTx, 20 min.
4. Wash 3 times in PBSTx, 5 min each.
5. Reduce animals, 10 min at 37°C.
6. Wash 3 times in PBSTx, then twice in PBS, 5 min per wash.
7. Bleach for 12 hr in 6% H2O2.1
8. Wash 3 times in PBSTx.
Immunolabeling:
1. Block 16 hr.
2. Incubate in mAb 3F11 (1:10 in blocking solution), overnight at 4°C.
3. Rinse in PBSTx, then wash 6 times, 1 hr each, in PBSTx.
4. Re-block 1 hr.
5. Incubate in goat anti-mouse HRP 1:250 plus DAPI 1 μg/ml, overnight at 4°C.
6. Rinse in PBSTx, then wash 6 times, 1 hr each, in PBSTx.
7. Wash 2 times in PBSTw, 5 min each.
8. TSA, 10 min.
9. Wash 3 times in PBSTx, 10 min each, then overnight.
10. Final rinse in PBSTx, then mount in Vectashield.
Note:
1 Optionally,
store unbleached animals overnight at 4°C prior to bleaching.
17
Forsthoefel DJ, Waters FA, and Newmark PA, 2014
594
595
596
597
598
599
600
601
602
603
604
605
606
607
608
609
610
611
612
613
614
615
616
617
618
619
620
621
622
623
624
625
626
627
628
629
630
631
632
633
634
635
Sample Protocol, mAb 2H3 (cryosections)
Processing:
1. Remove mucus in 2% HCl (3 min).
2. Fix in 4% formaldehyde/PBSTx, 20 min.1
3. Wash 3 times in PBSTx, 5 min each.
4. Incubate in 15% sucrose/1X PBS.
5. Incubate in 30% sucrose/1X PBS.
Cryosectioning:
1. Mount animals in tissue freezing medium.
2. Cryosection at 20 μm.
3. Store slides at -80°C.
Rehydration and AR:
1. Warm slides to RT, rehydrate in 1X PBS (3 x 5-10 min each).
2. Equilibrate in AR solution, 5 min, then microwave slides in AR solution, 10 min.
3. Cool gradually to RT, equilibrate in PBSTx.
Immunolabeling:
1. Block 30 min.
2. Incubate in mAb 2H3 (1:10 in blocking solution), 2 hr at RT.
3. PBSTx washes – rinses to remove coverslip and excess mAb, then 3 x 10 min.
4. Incubate in goat anti-mouse HRP 1:250 plus DAPI 1 μg/ml, 2 hr at RT.
5. PBSTx washes – rinses to remove coverslip and excess mAb, then 3 x 10 min.
6. PBSTw washes – 2 x 5 min.
7. TSA, 10 min.
8. PBSTx washes – rinses to remove coverslip and excess tyramide, then 3 x 10
min.
9. Mount in Vectashield.
Note:
1
Use freshly diluted 16% formaldehyde from Ted Pella.
18
Forsthoefel DJ, Waters FA, and Newmark PA, 2014
636
637
638
639
640
641
642
643
644
645
646
Optimal protocols for selected mAbs (whole animals)
Clone
Dilutiona
Relaxation/
Mucus Removal
Fixation
2G4
1:10
Mg; 2% HCl (3 min)
methacarn (20 min)
3F11
1:10
Mg; 2% HCl (3 min)
4% FA (20 min);
reduction
3G9
1:4
Mg; 2% HCl (3 min)
methacarn (20 min)
4D2
1:2*
Mg; 2% HCl (3 min)
methacarn (20 min)
1E12b
1:2*
Mg; 2% HCl (3 min)
methacarn (20 min)
1H8
1:2*
Mg; 7.5% NAc (7 min)
4% FA (20 min)
2C11
1:100
Mg; 7.5% NAc (7 min)
4% FA (20 min)
2D2
1:10
Mg; 7.5% NAc (7 min)
4% FA (20 min)
2H3
1:10
-/+ Mg;
7.5% NAc (7 min)
4% FA (20 min)
3G7
1:2*
2% HCl (3 min)
4% FA (20 min)
3H3
1:10
+/- Mg; 2% HCl (3 min)
methacarn (20 min)
a
Bleach
6% H2O2/
MeOH (12-16 hr)
6% H2O2/
PBS (12 hr)
6% H2O2/
MeOH (12 hr)**
6% H2O2/
MeOH (12-16 hr)**
6% H2O2/
MeOH (12-16 hr)**
6% H2O2/
MeOH (12-16 hr)**
6% H2O2/
MeOH (12-16 hr)
6% H2O2/
MeOH (12-16 hr)
6% H2O2/
MeOH (12-16 hr)**
6% H2O2/
MeOH (12-16 hr)**
6% H2O2/
MeOH (12-16 hr)**
Highest dilution tested, but may vary for unique batches of supernatant.
protocol to visualize intestinal nuclei.
* Dilution was not optimized.
** PBS bleaching was not tested.
b Optimal
19
Forsthoefel DJ, Waters FA, and Newmark PA, 2014
647
648
649
650
651
652
653
654
655
656
Optimal protocols for selected mAbs (cryosections)
Clone
Dilutiona
Mucus Removal
Fixation
Bleachb
AR
2G4
1:10
4% FA (20 min)
1:10
2C11
2D2
2H3
1:50
1:10
1:10
no
no (with HCl)
-oryes (with NAc)
yes
no
no
optional
3F11
3H3
1:10
2% HCl (3 min)
2% HCl (3 min)
-or7.5% NAc (7 min)
2% HCl (3 min)
2% HCl (3 min)
2% HCl (3 min)*
2% HCl (3 min)
-or7.5% NAc (7 min)
optional
optional
4% FA (20 min)
4% FA (20 min)
4% FA (20 min)
4% FA (20 min)
4% FA (20 min)
-ormethacarn (20 min)
a
Highest dilution tested, but may vary for unique batches of supernatant.
Bleaching (if specified) was performed in 6% H2O2/PBS (12 hr).
c Best protocol for 2H3 labeling of visceral muscle.
b
20
no
no
no
yes
Forsthoefel DJ, Waters FA, and Newmark PA, 2014
657
658
659
660
661
662
663
664
665
666
667
668
669
670
671
672
673
674
675
676
677
678
679
680
681
682
683
684
685
686
687
688
689
690
691
692
693
694
695
696
697
698
699
700
701
702
Working Solutions
7.5% NAc/PBS: Dissolve 7.5 g N-Acetyl-L-cysteine (Sigma) into 10 ml 1X PBS.
2% HCl: Dilute 0.556 ml 36% HCl into 9.5 ml ultra-pure water (1:18 dilution).
Methacarn: For 10 ml, mix 6 ml methanol, 3 ml chloroform, and 1 ml glacial
acetic acid. Methacarn solution can be stored for 1-2 weeks at 4°C.
4% formaldehyde solution: For whole animal fixation, dilute 1 ml 36.5%
methanol-stabilized formaldehyde solution (EMD/Millipore) into 8 ml PBSTx. For
animals that will be cryosectioned, mix 10 ml 16% formaldehyde (Ted Pella), 4 ml
10X PBS, and 1.2 ml 10% Triton X-100. Store at 4°C for <2 weeks.
PBSTx: 1X PBS plus 0.3% Triton X-100
Reduction solution: 50 mM DTT, 1% NP-40, 0.5% SDS, 1X PBS
6% H2O2/methanol: Dilute 2 ml 30% H2O2 into 8 ml methanol.
6% H2O2/PBS: Dilute 2 ml 30% H2O2 into 8 ml 1X PBS.
Proteinase K solution: 10 μg/ml proteinase K (Invitrogen) in PBSTx plus 0.1%
SDS
AR solution: 10 mM sodium citrate, pH 6.0 (can dilute from filter sterilized 100
mM stock)
Blocking solution: 0.6% (w/v) IgG-free BSA (Jackson), 0.45% (v/v) fish gelatin
(Sigma), 1X PBS, 0.3% Triton X-100
4.5% fish gelatin stock solution: Warm 45% fish gelatin to 50°C, dilute 1:10 in
ultra-pure water, store at 4°C, re-warm prior to further dilution.
PBSTw: 1X PBS plus 0.01% Tween-20
0.5% H2O2: 30% H2O2 stock diluted 1:60 into PBSTw.
Tyramide solution: FITC-Tyramide diluted 1:1500 plus 0.5% H2O2 diluted 1:100
(final concentration 0.005%) into PBSTw (e.g., 1 μl FITC-Tyramide, 15 μl 0.5%
0.5% H2O2, 1.5 ml PBSTw). FITC-Tyramide was synthesized as in [5].
15% sucrose solution: 15% sucrose (w/v) in 1X PBS.
30% sucrose solution: 30% sucrose (w/v) in 1X PBS.
21
Forsthoefel DJ, Waters FA, and Newmark PA, 2014
703
704
705
Reagents and Equipment
Description
Manufacturer
Catalog Number/ID
RPI
RPI
JT Baker/VWR
Sigma-Aldrich
EMD/Millipore
Sigma
Sigma
Invitrogen
Fisher
125505
121002
9535-03
A7250
FX0410-5
18505
H1009
25530-049
S279
Jackson ImmunoResearch
Sigma-Aldrich
001-000-162
G7765
Jackson ImmunoResearch
115-035-044
Sigma-Aldrich
Vector Laboratories
D9542
H-1000
Ted Pella
Ted Pella
Fisher
Ted Pella
Sigma-Aldrich
Sigma-Aldrich
106
27209
12-550-15
2108
G2500
C337
Ted Pella
Ted Pella
Fisher/Gold Seal
Ted Pella
US Plastic
432-1
21029
3317
21096
53669
Processing:
Glass Vials (small)
Glass Vials (large)
HCl
N-Acetyl-L-cysteine
36.5% formaldehyde
16% formaldehyde
30% H2O2
Proteinase K solution
Sodium Citrate
Labeling:
IgG-free BSA
45% fish gelatin
Peroxidase-conjugated goat antimouse IgG+IgM (H+L)
DAPI
Vectashield
Cryosectioning:
Pelco 106 embedding molds
TBS Tissue Freezing Medium
SuperFrost PLUS slides
Handy Mini Slide Box
Gelatin
Chromium (III) Potassium Sulfate
Labeling cryosections:
Glass staining jars
Polypropylene Coplin jars
22x50 mm No. 1 glass coverslips
Slide Staining Jar (low volume)
“Tuff Tainer” Slide Staining Box
706
707
708
709
710
711
712
713
22
Forsthoefel DJ, Waters FA, and Newmark PA, 2014
714
715
716
717
718
719
References for Additional File 2
1.
Gurley KA, Rink JC, Sánchez Alvarado A: β-catenin defines head
versus tail identity during planarian regeneration and homeostasis.
Science 2008, 319(5861):323-327.
720
721
722
2.
Umesono Y, Watanabe K, Agata K: A planarian orthopedia homolog is
specifically expressed in the branch region of both the mature and
regenerating brain. Dev Growth Differ 1997, 39(6):723-727.
723
724
725
3.
Pearson BJ, Eisenhoffer GT, Gurley KA, Rink JC, Miller DE, Sánchez
Alvarado A: Formaldehyde-based whole-mount in situ hybridization
method for planarians. Dev Dyn 2009, 238(2):443-450.
726
727
728
4.
Wang Y, Zayas RM, Guo T, Newmark PA: nanos function is essential
for development and regeneration of planarian germ cells. Proc Natl
Acad Sci U S A 2007, 104(14):5901-5906.
729
730
731
732
733
5.
King RS, Newmark PA: In situ hybridization protocol for enhanced
detection of gene expression in the planarian Schmidtea
mediterranea. BMC Dev Biol 2013, 13:8.
23