High-Throughput, Multi-Image Cryohistology of Mineralized Tissues

Summary

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.

Abstract

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.

Introduction

Biological research often requires efficient phenotyping, which is frequently associated with some sort of histological analysis^1-3. 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....

Protocol

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 limbs with joints at consistent flexion, internal rotation, and external rotation angles¹¹. Typically, fix whole mouse limbs for 1 - 3 days.

Results

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.......

Discussion

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 .......

Materials

List of materials used in this article

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.