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Biology

High-Throughput, Multi-Image Cryohistology of Mineralized Tissues

Published: September 14th, 2016

DOI:

10.3791/54468

1Department of Reconstructive Sciences, University of Connecticut Health Center, 2Department of Computer Science and Engineering, University of Connecticut, 3Department of Orthopaedic Surgery, University of Connecticut Health Center, 4Department of Orthopaedics, University of Rochester

In this manuscript, we present a high-throughput, semi-automated cryohistology platform to produce aligned composite images of multiple response measures from several rounds of fluorescent imaging on frozen sections 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.

Biological research often requires efficient phenotyping, which is frequently associated with some sort of histological analysis1-3. This need is even more evident with the ambitious projects to disrupt each gene in the mouse genome4. These histological analyses can range from assessing cell morphology and/or anatomical features to mapping expression of specific genes or proteins to individual cells. In fact, one of the fundamental contributions of histology to the field of genomics is the ability to associate a specific molecular signal to a specific region or cell type.

Traditional methods of histology, especially fo....

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The University of Connecticut Health Center institutional animal care and use committee approved all animal procedures.

1. Fixation and Embedding

  1. Euthanize the animal via CO2 asphyxiation or other approved methods.
  2. Harvest the tissue of interest (e.g., limb, vertebrae, etc.) and place in 10% neutral buffered formalin at 4 °C until properly fixed. Take special care to maintain consistent anatomical placement prior to fixation. For instance, fix lim.......

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A General Workflow for the High-Throughput, Multi-Image Cryohistology

Figure 1 represents the general workflow used for this technique. It includes several steps from fixation through several rounds of imaging and finally image alignment/analysis. The process can take as little as a week to go from sample fixation through 4 rounds of imaging, which is less time than it takes to decalcify these t.......

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Here we have presented a detailed cryohistology protocol to co-localize and quantify several biological measures by aligning images from multiple rounds of staining/imaging on a single section. The method outlined using the cryotape is especially useful as it maintains the morphology of difficult to section tissue (e.g., mineralized bone and cartilage). In addition, the sectioned tissue is adhered firmly to the glass slide, allowing for multiple rounds of staining/imaging of the same section; unlike traditional .......

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The authors would like to acknowledge the following funding sources: NIH R01-AR063702, R21-AR064941, K99-AR067283, and T90-DE021989.

....

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Name Company Catalog Number Comments
10% neutral buffered formalin Sigma Aldrich HT501128-4L Multiple suppliers available. Toxic. Can be substituted with 4% paraformaldehyde.
Sucrose Sigma Aldrich S9378 Multiple suppliers available.
PBS Sigma Aldrich P5368 Multiple suppliers available.
Cryomolds Fisher Scientific Fisherbrand #22-363-554 Different sizes can be used depending on tissue
Cryomatrix Thermo Scientific 6769006 Can be substitituted with other cryomatrices. However, PVA/PEG-based resins have worked best in our hands.
2-methyl-butane Sigma Aldrich M32631 Multiple suppliers available.
Cryostat Leica Biosystems 3050s Can be substituted with other brands/models.
Specimen disc Leica Biosystems 14037008587 Can be substituted with other brands/models.
Cryostat blades Thermo Scientific 3051835 Can be substituted with other brands/models.
Cryotape Section Lab Cryofilm 2C
Roller Electron Microscopy Sciences 62800-46 Can be substituted with other brands/models.
Plastic microscope slides Electron Microscopy Sciences 71890-01 Can be substituted with other brands/models.
Glass microscope slides Thermo Scientific 3051 Can be substituted with other brands/models.
Norland Optical Adhesive, 61 Norland Optical Norland Optical Adhesive, 61
UV Black Light General Electric F15T8-BLB
Glacial acetic acid Sigma Aldrich ARK2183 Multiple suppliers available.
Chitosan Sigma Aldrich C3646 Multiple suppliers available.
InSpeck red microscopheres ThermoFisher Scientific I-14787
InSpeck green microspheres ThermoFisher Scientific I-14785
Calcein Blue Sigma Aldrich M1255 Multiple suppliers available.
Calcein Sigma Aldrich C0875  Multiple suppliers available.
Alizarin complexone Sigma Aldrich A3882  Multiple suppliers available.
Demeclocycline Sigma Aldrich D6140  Multiple suppliers available.
NaHCO3 Sigma Aldrich S5761 Multiple suppliers available.
Glycerol Sigma Aldrich G5516 Multiple suppliers available.
ELF 97 yellow fluorescent acid phosphatase substrate ThermoFisher Scientific E-6588
DAPI ThermoFisher Scientific 62247 Multiple suppliers available. Can be substituted with Hoechst 33342 or other nuclear dyes.
TO-PRO-3 (Cy5 nuclear counterstain) ThermoFisher Scientific T3605
Propidium Iodide ThermoFisher Scientific R37108 Multiple suppliers available.
Sodium acetate anhydrous Sigma Aldrich S2889 Multiple suppliers available.
sodium tartrate dibasic dihydrate  Sigma Aldrich T6521 Multiple suppliers available.
Sodium nitrite Sigma Aldrich S2252 Multiple suppliers available.
Tris Sigma Aldrich 15504 Multiple suppliers available.
MgCl2 hexahydrate Sigma Aldrich M9272 Multiple suppliers available.
NaCl Sigma Aldrich S7653 Multiple suppliers available.
Fast Red TR Salt Sigma Aldrich F8764 Multiple suppliers available. Can also be substituted with other substrate kits such as Vector Blue (Vector Laboratories, Cat# SK-5300)
Naphthol AS-MX  Sigma Aldrich N4875 Multiple suppliers available.
N,N dimethylformamide Sigma Aldrich D158550 Multiple suppliers available.
Toluidine blue O Sigma Aldrich T3260 Multiple suppliers available.
Axio Scan.Z1 Carl Zeiss AG Axio Scan.Z1 Other linear or tiled scanners may also be used.
DAPI Filter Set Chroma Technology Corp. 49000
CFP Filter Set Chroma Technology Corp. 49001
GFP Filter Set Chroma Technology Corp. 49020
YFP Filter Set Chroma Technology Corp. 49003
Custom yellow (ELF 97) Filter Set Chroma Technology Corp. custom; HQ409sp, HQ555/30m, 425dxcr
TRITC Filter Set Chroma Technology Corp. 49004
Cy5 Filter Set Chroma Technology Corp. 49009
CryoJane Tape Transfer System Electron Microscopy Sciences 62800-10 Multiple suppliers available.
CryoJane Tape Windows Electron Microscopy Sciences 62800-72 Multiple suppliers available.
CryoJane Adhesive Slides Electron Microscopy Sciences 62800-4X Multiple suppliers available.

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