De alto rendimento, Multi-Imagem Cryohistology de mineralizadas tecidos

Resumo

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.

Resumo

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.

Introdução

Pesquisa biológica, muitas vezes requer fenotipagem eficiente, o que é frequentemente associada com algum tipo de análise histológica ^1-3. Esta necessidade é ainda mais evidente com os projectos ambiciosos para interromper cada gene no genoma do rato ^4. Estas análises histológicas pode variar de avaliar a morfologia celular e / ou características anatómicas a expressão de mapeamento de genes específicos ou proteínas para células individuais. Na verdade, uma das contribuições fundamentais de histologia para o campo da genômica é a capacidade de associar um sinal molecular específica para uma região ou célula tipo específico.

Protocolo

A Universidade do comitê de cuidados com os animais institucional e uso Connecticut Centro de Saúde aprovou todos os procedimentos com animais.

1. Fixação and Embedding

1. Eutanásia do animal através de CO ₂ asfixia ou outros métodos aprovados.
2. Colheita do tecido de interesse (por exemplo, membro, vértebras, etc.) e colocar em 10% de formalina tamponada neutra a 4 ° C até ser adequadamente fixo. Tome especial cuidado para manter a colocação anatômica consistente antes da fixação. Por exemplo, corrigir membros com juntas em flexão consistente, rotação interna e rotação externa ângulos ^11. No

Resultados

Um fluxo de trabalho geral para o Alto Rendimento, Multi-Imagem Cryohistology

A Figura 1 representa o fluxo de trabalho geral utilizado para esta técnica. Ele inclui várias etapas de fixação através de várias rodadas de imagem e, finalmente, de alinhamento de imagem / análise. O processo pode demorar tão pouco como uma semana para ir de fixação de amostra através de 4 rodadas de ima...

Discussão

Aqui nós apresentamos um protocolo cryohistology detalhada para co-localizar e quantificar várias medidas biológicas, alinhando imagens de várias rodadas de coloração / imagens em uma única seção. O método descrito utilizando o cryotape é especialmente útil uma vez que mantém a morfologia do tecido difícil de secção (por exemplo, osso mineralizado e cartilagem). Além disso, o tecido seccionados é aderida firmemente para a lâmina de vidro, para permitir que vários ciclos de coloração / imag...

Materiais

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.