Large-scale tissue clearing (PACT): Technical evaluation and new perspectives in immunofluorescence, histology, and ultrastructure.
Novel techniques, like CLARITY and PACT, render large tissue specimens transparent and thereby suitable for microscopic analysis. We used these techniques to evaluate their potential in the intestine as an exemplary organ with a complex tissue composition. Immunohistochemistry, light sheet-, and confocal scanning-microscopy enabled us to follow complex three-dimensional structures, like nerve fibers, vessels, and epithelial barriers throughout the entire organ. Moreover, in a systematic electron microscopic study, we analyzed the morphology and preservation of tissue on ultrastructural level during the clearing process. We also connect tissue clearing with classical histology and demonstrate that cleared tissues can be stained with Hematoxylin-Eosin and Heidenhain's Azan stain, suggesting potential use in histopathology. These experiments showed that a neutral pH during the clearing process results in much better preservation of tissue ultrastructure and standard stainability. Volume changes of specimens were monitored and quantified during the course of the protocol. Additionally, we employed the technique to visualize the enteric nervous system and the epithelial barrier in post mortem human gut preparations. Our data show the high potential of tissue clearing throughout different tissue types supporting its usefulness in research and diagnosis, and contribute to the technical discussion of ultrastructural tissue-retention.
Institute of Clinical Anatomy and Cell Analysis, University of Tübingen, Tübingen, Germany.
Peter H
PH
Neckel
Institute of Clinical Anatomy and Cell Analysis, University of Tübingen, Tübingen, Germany.
Ulrich
U
Mattheus
Institute of Clinical Anatomy and Cell Analysis, University of Tübingen, Tübingen, Germany.
Bernhard
B
Hirt
Institute of Clinical Anatomy and Cell Analysis, University of Tübingen, Tübingen, Germany.
Lothar
L
Just
Institute of Clinical Anatomy and Cell Analysis, University of Tübingen, Tübingen, Germany.
Andreas F
AF
Mack
Nat Protoc. 2015 Nov;10(11):1709-27
26448360
Exp Neurol. 2013 Apr;242:57-64
23124097
PLoS One. 2016 Jun 30;11(6):e0158628
27359336
Cell. 2014 Aug 14;158(4):945-58
25088144
Sci Rep. 2015 May 27;5:10640
26013317
BMC Dev Biol. 2014 Dec 21;14:48
25528649
Adv Exp Med Biol. 2016;891:193-9
27379646
Nat Neurosci. 2011 Aug 30;14(11):1481-8
21878933
Cell. 2014 Nov 6;159(4):896-910
25417164
Sci Rep. 2016 Jan 11;6:18631
26750588
Nat Protoc. 2012 Nov;7(11):1983-95
23060243
Dig Dis Sci. 2014 Jul;59(7):1529-34
24744180
Cell. 2015 Dec 3;163(6):1500-14
26638076
Cell. 2015 Jul 16;162(2):246-57
26186186
Nat Methods. 2013 Jun;10(6):508-13
23722210
Neurogastroenterol Motil. 2011 Feb;23(2):115-24
21175997
Nat Neurosci. 2013 Aug;16(8):1154-61
23792946
Development. 2013 Mar;140(6):1364-8
23444362
eNeuro. 2015 May 25;2(3):null
26464982
Nature. 2013 May 16;497(7449):332-7
23575631
J Comp Neurol. 2010 Nov 1;518(21):4277-87
20853506
Nat Protoc. 2014 Jul;9(7):1682-97
24945384
Neurogastroenterol Motil. 2013 May;25(5):e324-38
23495930
Ann Anat. 1999 Jul;181(4):327-37
10427369
Neuroscientist. 2013 Apr;19(2):195-207
22645111
Am J Physiol Gastrointest Liver Physiol. 2013 Jan 1;304(1):G1-11
23086917