High-Throughput, Multi-Image Cryohistology of Mineralized Tissues.
There is an increasing need for efficient phenotyping and histopathology of a variety of tissues. This phenotyping need is evident with the ambitious projects to disrupt every gene in the mouse genome. The research community needs rapid and inexpensive means to phenotype tissues via histology. Histological analyses of skeletal tissues are often time consuming and semi-quantitative at best, regularly requiring subjective interpretation of slides from trained individuals. Here, we present a cryohistological paradigm for efficient and inexpensive phenotyping of mineralized tissues. First, we present a novel method of tape-stabilized cryosectioning that preserves the morphology of mineralized tissues. These sections are then adhered rigidly to glass slides and imaged repeatedly over several rounds of staining. The resultant images are then aligned either manually or via computer software to yield composite stacks of several layered images. The protocol allows for co-localization of numerous molecular signals to specific cells within a given section. In addition, these fluorescent signals can be quantified objectively via computer software. This protocol overcomes many of the shortcomings associated with histology of mineralized tissues and can serve as a platform for high-throughput, high-content phenotyping of musculoskeletal tissues moving forward.
Department of Reconstructive Sciences, University of Connecticut Health Center; dyment@uchc.edu.
Nathaniel A
NA
Dyment
Department of Reconstructive Sciences, University of Connecticut Health Center.
Xi
X
Jiang
Department of Reconstructive Sciences, University of Connecticut Health Center.
Li
L
Chen
Department of Computer Science and Engineering, University of Connecticut.
Seung-Hyun
SH
Hong
Department of Orthopaedic Surgery, University of Connecticut Health Center.
Douglas J
DJ
Adams
Department of Orthopaedics, University of Rochester.
Cheryl
C
Ackert-Bicknell
Department of Computer Science and Engineering, University of Connecticut.
Dong-Guk
DG
Shin
Department of Reconstructive Sciences, University of Connecticut Health Center; drowe@uchc.edu.
David W
DW
Rowe
D000818:Animals; D015925:Cryopreservation; D006652:Histological Techniques; D006801:Humans; D051379:Mice; D008903:Minerals; D010641:Phenotype; D012984:Software
D008903:Minerals
J Orthop Res. 2015 Nov;33(11):1693-703
25982892
Osteoarthritis Cartilage. 2016 Feb;24(2):335-44
26362410
J Bone Miner Res. 2014;29(5):1283-94
24190076
Stem Cells. 2012 Feb;30(2):187-96
22083974
J Orthop Res. 2010 Oct;28(10):1338-47
20839319
J Tissue Sci Eng. 2012 Dec 24;Suppl 1:004
25019033
Dis Model Mech. 2012 Jan;5(1):19-25
22028326
PLoS Genet. 2006 Apr;2(4):e61
16683035
Microsc Res Tech. 2001 May 1;53(3):239-40
11301500
Dis Model Mech. 2014 May;7(5):515-24
24652767
Dev Biol. 2015 Sep 1;405(1):96-107
26141957
J Orthop Res. 2015 Jul;33(7):948-56
25639792
Osteoarthritis Cartilage. 2015 Jun;23 (6):996-1006
25680653
Arch Histol Cytol. 2003 May;66(2):123-43
12846553
Bone. 2016 Mar;84:69-77
26721734
J Orthop Res. 2015 Aug;33(8):1142-51
25807894
J Histochem Cytochem. 2005 May;53(5):593-602
15872052
Mamm Genome. 2012 Oct;23(9-10):600-10
22961258