Name: 세미 두께 뇌 조각의 고해상도 형광 이미징에 대한 빠른 접근 (동영상) | JoVE Journal
Uploaded: 2011-07-26T12:00:00.000+00:00
Duration: 4 min 35 s
Description: 배우세요 세미 두께 뇌 조각에서 형광 뉴런을 고해상도로 시각화하는 방법을. 뇌 조직을 고정하고 절단한 후 광학적 투명화를 통해 개별 세포와 신경망을 표준 현미경으로 관찰할 수 있습니다.

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This work was funded by support through the McNair Foundation, NARSAD, and NINDS grant R00NS064171-03.

1. In situ brain fixation
*Prepare a 10 ml syringe (28 gauge needle) filled with phosphate buffered saline (PBS). 
*Prepare a 10 ml syringe (28 gauge needle) filled with 4% paraformaldehyde (PFA) in PBS. Reserve an additional 5-10 ml of PFA/PBS for post fixation.

<ol>
<li>Inject experimental mouse intraperitoneally with a lethal dose of Avertin or Nembutal.</li>
<li>Once the mouse is sedated, wet the abdomen with ethanol and secure the mouse to the bottom of a tray layered with cork, wax, or silicone elastomer using dissection pins. With abdomen facing up, secure the four paws to the surface - spreading them as wide as possible.</li>
<li>Grab the skin with forceps at the level of the sternum, and cut crosswise to expose the liver. Cut laterally and then up through the ribs and diaphragm. Remove the rib tissue flap and continue cutting until the heart is accessible.</li>
<li>Pierce or snip the right atrium to drain circulated blood.</li>
<li>Flush residual blood from circulatory system by perfusion of PBS through left ventricle.</li>
<li> Accessing the same needle hole, fix whole mouse by subsequent perfusion of PBS/PFA through left ventricle.</li>
</ol>
2. Dissection and brain extraction 
<ol>
<li>Remove the skin from head, neck, and cranium by cutting around ear canal and eye orbits, followed by pulling skin anteriorly to expose the cranium. 
*See Figure 2 for an illustration of steps 2.2) to 2.5)</li>
<li>Using a bone scissors, cut crosswise from eye the socket through the nasal turbinate bones. Do this for each side. The crosscut should be anterior to the eyes and olfactory bulbs.</li>
<li>Cut lengthwise from each ear canal to eye socket.</li>
<li>Using a small dissection scissors, cut the occipital bone plate overlying the cerebellum from one ear canal to the other.</li>
<li>With the same dissection scissors, cut anteriorly along the midline to the level of the olfactory bulbs.</li>
<li>Using forceps, remove each bone plate from the brain, taking care to remove any connective tissue so that the brain remains intact when lifting off the bone.</li>
<li>Snip optic and cranial nerves, cut the top cervical vertebrae, and remove the brain into fixative.</li>
<li>Post-fix for 1-2 hrs at 4° C. Wash 3 times 15 min each in PBS.</li>
</ol>
3. Agarose embedding
<ol>
<li>Melt 2% high-strength, low-gel point agarose type I-B (Sigma catalog number: A0576) in PBS saline with microwave oven.</li>
<li>Transfer molten agarose to a test tube. Place test tube in a warm incubator or water bath (40°-41° C) until temperature equilibration.</li>
<li>Glue fixed brain tissue onto the plunger of the specimen syringe with cyanoacrylate glue (Figure 3A). Brush a thin layer of saturated sucrose-PBS solution on the surface of the brain tissue to facilitate the later release of the agarose ring from the brain slices.</li>
<li>Draw down the plunger with brain tissue into the plunger housing (Figure 3B).</li>
<li>Pipette or pour warm agarose into the plunger housing, covering the brain tissue. Avoid trapping air bubbles into the agarose. Air bubbles in the agarose will compromise the slice quality.</li> 
<li>Clamp the specimen syringe with a cold (0° to -25° C) chilling block for 1 minute to set the agarose (Figure 3C).</li> 
</ol>
4. Sectioning brain slice with the Compresstome
<ol>
<li>Assemble the specimen syringe containing the brain tissue into the Compresstome (Figure 3D).</li>
<li>Align the edge of the razor blade closely with the outlet of the specimen syringe (Figure 3E).</li>
<li>Fill up the buffer tank with PBS solution.</li>
<li>Rotate the micrometer one slice thickness forward to extrude the agarose-brain tissue block out of the specimen syringe.</li>
<li>Press the “start” button on the control unit of the Compresstome to initiate the sectioning process.</li> 
<li>Repeat steps 4.4) to 4.5) until desired slices are obtained at determine thickness.</li> 
<li>Harvest brain slices with brush or pipette to vials or staining wells filled with PBS (Figure 3F).</li>
</ol>
5. Optical Clearing
<ol>
<li>Prepare a 75% vol:vol solution of glycerol:PBS.</li>
<li>Pour (or pipette) off half the volume of PBS wash from the collected brain slices.</li>
<li>Add back the volume removed with glycerol/PBS.</li>
<li>Allow to equilibrate with gentle mixing at 4° C until the slices sink.</li>
<li>Repeat steps 6.2) to 6.4) until a final glycerol concentration reaches 75%.</li>
<li> Replace solution with 90% glycerol in PBS and allow to equilibrate. 
*Note: Take care not to introduce air bubbles while adding glycerol solutions. Pour solutions slowly and mix gently, since air bubbles will interfere with uniform equilibration and will be carried over to slide mounting.</li>
</ol>
6. Slice mounting for imaging
<ol>
<li>Cut double-sided seal adhesive (SA-S-1L, Grace Bio-Labs) into an open rectangle to fit the viewable area of a micro slide (Superfrost Plus, 25 X 75 X 1 mm, cat. # 48311-703, VWR) with sides 2-3 mm wide.</li>
<li>Place brain slices inside the center of the adhesive tape rectangle using a pipette or paintbrush. Remove excess glycerol with gentle aspiration.</li>
<li>Arrange brain slices on slide in desired order with a paintbrush.</li>
<li>Gently lower cover glass (24 X 60 mm, cat. # 48383-139, VWR) onto the adhesive tape, taking care not to introduce air bubbles. Gently apply pressure onto cover glass to ensure contact with adhesive tape.</li>
<li>Aspirate excess glycerol from edges between slide and coverglass.</li>
<li>Seal slide and coverslip using clear nail polish.</li>
<li>Allow to dry before imaging, or store long-term in slide boxes at 4° C.</li>
</ol>
7. Representative Results: 
Processing, imaging, and analyzing fluorescently labeled brain tissue have become indispensible to the study of neurobiology. Many of these investigations require sophisticated genetic manipulations to obtain reporter expression in targeted neuronal subsets, followed by both low- and high-resolution image analyses. Often experimental and technical limitations make it impossible to obtain these types of data from the same animal, or in a rapid manner. Preparation of optically-cleared, semi-thick brain sections for fluorescent imaging aids in this challenge. An example of an intact thick brain slice labeled with a genetically modified rabies virus engineered to express EGFP, and imaged using epifluorescent and confocal microscopy is shown in Figure 4 and Figure 5 respectively. For epifluorescent imaging we used a Leica M205 FA, and for confocal image acquisition we used a Zeiss LSM 510. Due to the relative simplicity of the attached protocol, this method is capable of yielding useful image data from tissue expressing fluorescent reporters with a turnaround time of less than one day, and is compatible with both low- and high-resolution light microscopy. 
If the investigator chooses to incorporate additional postmortem labeling methods, immunohistochemical staining, or make thinner sections, the protocol lengthens accordingly. However, the method described above represents a simple and relatively high-throughput method for screening patterns of vital reporter expression in intact brain. 
<img src="/files/ftp_upload/2807/2807fig1.jpg" alt="Brain dissection process diagram; perfusion to slice mounting for optical clearing." data-altupdatedat="1788235205000" data-alt="Figure 1"> Figure 1. Flow chart diagraming the fixation, dissection, slicing, clearing, and mounting procedure of semi-thick brain slices.
<img src="/files/ftp_upload/2807/2807fig2.jpg" alt="Skull diagram with labeled lines 2.2 to 2.5, illustrating anatomical measurement markers." data-altupdatedat="1788235205000" data-alt="Figure 2"> Figure 2. Diagram of sequential cutting steps to extract the intact mouse brain. 
<img src="/files/ftp_upload/2807/2807fig3.jpg" alt="Static equilibrium in metal testing process using force application; includes equipment and setup." data-altupdatedat="1788235205000" data-alt="Figure 3"> Figure 3. Steps to mount and slice brain tissue using the Compresstome. A) Placement of brain tissue onto cutting plunger using superglue. B) Drawing down the mounted brain tissue into the plunger for agarose embedding. C) Solidifying an agarose brain plug using a chilled compression block. D) Insertion of agarose brain plug and plunger into Compresstome cutting chamber. E) Alignment of razorblade to the plunger device. F) Cutting and collection of brain slices into Compresstome buffer chamber.
<img src="/files/ftp_upload/2807/2807fig4.jpg" alt="Brain fluorescence microscopy; green signal imaging; neuronal activity mapping; optical analysis." data-altupdatedat="1788235205000" data-alt="Figure 4"> Figure 4. Light microscopy images of a thick slice from glycerol-cleared brain tissue through the frontal cortex expressing enhanced green fluorescent protein (EGFP). A) Coronal slice through a mouse brain (200 um thick) labeled with a viral vector expressing EGFP and imaged at low resolution using an epifluorescent stereoscope. Scale bar, 2 mm. 
<img src="/files/ftp_upload/2807/2807fig5.jpg" alt="Neuronal cells fluorescent microscopy image, highlighting cell morphology and network structure." data-altupdatedat="1788235205000" data-alt="Figure 5"> Figure 5. High magnification image of fluorescently labeled layer 5/6 cortical neurons in a cleared thick brain slice. A) High resolution confocal image of a maximum projection Z-stack (150 um thick) through the region highlighted in (Figure 4). Scale bar, 25 um.

<ol><li>Pfister, H., Lichtman, J., Reid, C. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aPfister%2C+H+%22The+Connectome+Project%22">The Connectome Project</a>. , (2009).</li>
<li>Briggman, K. L., Denk, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Towards+neural+circuit+reconstruction+with+volume+electron+microscopy+techniques%5BTitle%5D)+AND+%22Curr+Opin+Neurobiol%22%5BJournal%5D)">Towards neural circuit reconstruction with volume electron microscopy techniques.</a> Curr Opin Neurobiol. 16, 562-570 (2006).</li>
<li>Micheva, K. D., Smith, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Array+tomography%3A+a+new+tool+for+imaging+the+molecular+architecture+and+ultrastructure+of+neural+circuits%5BTitle%5D)+AND+%22Neuron%22%5BJournal%5D)">Array tomography: a new tool for imaging the molecular architecture and ultrastructure of neural circuits.</a> Neuron. 55, 25-36 (2007).</li>
<li>Arenkiel, B. R., Ehlers, <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Molecular+genetic+and+imaging+technologies+for+circuit+based+neuroanatomy%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">Molecular genetic and imaging technologies for circuit based neuroanatomy.</a> Nature. 461, 900-907 (2009).</li>
<li>Capecchi, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Altering+the+genome+by+homologous+recombination%5BTitle%5D)+AND+%22Science%22%5BJournal%5D)">Altering the genome by homologous recombination.</a> Science. 244, 1288-1292 (1989).</li>
<li>Luo, L., Callaway, E. M., Svoboda, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Genetic+dissection+of+neural+circuits%5BTitle%5D)+AND+%22Neuron%22%5BJournal%5D)">Genetic dissection of neural circuits.</a> Neuron. 57, 634-660 (2008).</li>
<li>Shimomura, O., Johnson, F., Saiga, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Extraction%2C+purification%2C+and+properties+of+aequorin%2C+a+bioluminescent+protein+from+the+luminous+hydromedusan%2C+Aequorea%5BTitle%5D)+AND+%22J.+Cell+Comp+Physiol%22%5BJournal%5D)">Extraction, purification, and properties of aequorin, a bioluminescent protein from the luminous hydromedusan, Aequorea.</a> J. Cell Comp Physiol. 59, 223-239 (1962).</li>
<li>Chalfie, M., Tu, Y., Euskirchen, G., Ward, W., Prasher, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Gene+fluorescent+protein+as+a+marker+for+gene+expression%5BTitle%5D)+AND+%22Science%22%5BJournal%5D)">Gene fluorescent protein as a marker for gene expression.</a> Science. 263, 802-805 (1994).</li>
<li>Giepmans, B. N., Adams, S. R., Ellisman, M. H., Tsien, R. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+fluorescent+toolbox+for+assessing+protein+location+and+function%5BTitle%5D)+AND+%22Science%22%5BJournal%5D)">The fluorescent toolbox for assessing protein location and function.</a> Science. 312, 217-224 (2006).</li>
<li>Micheva, K. D., Smith, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Array+tomography%3A+a+new+tool+for+imaging+the+molecular+architecture+and+ultrastructure+of+neural+circuits%5BTitle%5D)+AND+%22Neuron%22%5BJournal%5D)">Array tomography: a new tool for imaging the molecular architecture and ultrastructure of neural circuits.</a> Neuron. 55, 25-36 (2007).</li>
<li>Svoboda, K., Yasuda, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Principles+of+two-photon+excitation+microscopy+and+its+applications+to+neuroscience%5BTitle%5D)+AND+%22Neuron%22%5BJournal%5D)">Principles of two-photon excitation microscopy and its applications to neuroscience.</a> Neuron. 50, 823-839 (2006).</li>
</ol>

Jian-Qiang Kong is an employee of Precisionary Instruments, Inc., that manufactures a product used in this article.

Given the widespread application of using fluorescent proteins to target neuronal subsets for investigation via light microscopy, the need to rapidly screen, image, and analyze neural networks within intact brain tissue has become invaluable. 
Technical advances in the development of user-friendly viral vectors, in vivo electroporation techniques, and genetically modified mouse strains now provides a seemingly unlimited source of labeled cell types to investigate. However, image anal...

<table><tbody><tr><td>bone scissors</td><td>F.S.T.</td><td>16044-10</td><td>-or equivalent</td></tr><tr><td>dissection scissors</td><td>F.S.T.</td><td>14084-08</td><td>-or equivalent</td></tr><tr><td>type I-B agarose</td><td>Sigma-Aldrich</td><td>A0576</td><td></td></tr><tr><td>Compresstome</td><td>Precisionary Instruments</td><td>VF-200</td><td>-other vibratomes are compatible </td></tr><tr><td>double sided adhesive</td><td>Grace Bio-Lab Inc.</td><td>SA-S-1L</td><td></td></tr><tr><td>Superfrost Plus slides</td><td>VWR international</td><td>48311-703</td><td></td></tr><tr><td>Cover glass</td><td>VWR international</td><td>48383-139</td><td></td></tr><tr><td>glycerol</td><td>EMD Millipore</td><td>GX0185-6</td><td>-or equivalent</td></tr></tbody></table>

a rapid approach to high-resolution fluorescence imaging in semi-thick brain slices

A Rapid Approach to High-Resolution Fluorescence Imaging in Semi-Thick Brain Slices

A fundamental goal to both basic and clinical neuroscience is to better understand the identities, molecular makeup, and patterns of connectivity that are characteristic to neurons in both normal and diseased brain. Towards this, a great deal of effort has been placed on building high-resolution neuroanatomical maps1-3. With the expansion of molecular genetics and advances in light microscopy has come the ability to query not only neuronal morphologies, but also the molecular and cellular makeup of individual neurons and their associated networks4. Major advances in the ability to mark and manipulate neurons through transgenic and gene targeting technologies in the rodent now allow investigators to 'program' neuronal subsets at will5-6. Arguably, one of the most influential contributions to contemporary neuroscience has been the discovery and cloning of genes encoding fluorescent proteins (FPs) in marine invertebrates7-8, alongside their subsequent engineering to yield an ever-expanding toolbox of vital reporters9. Exploiting cell type-specific promoter activity to drive targeted FP expression in discrete neuronal populations now affords neuroanatomical investigation with genetic precision.
Engineering FP expression in neurons has vastly improved our understanding of brain structure and function. However, imaging individual neurons and their associated networks in deep brain tissues, or in three dimensions, has remained a challenge. Due to high lipid content, nervous tissue is rather opaque and exhibits auto fluorescence. These inherent biophysical properties make it difficult to visualize and image fluorescently labelled neurons at high resolution using standard epifluorescent or confocal microscopy beyond depths of tens of microns. To circumvent this challenge investigators often employ serial thin-section imaging and reconstruction methods10, or 2-photon laser scanning microscopy11. Current drawbacks to these approaches are the associated labor-intensive tissue preparation, or cost-prohibitive instrumentation respectively.
Here, we present a relatively rapid and simple method to visualize fluorescently labelled cells in fixed semi-thick mouse brain slices by optical clearing and imaging. In the attached protocol we describe the methods of: 1) fixing brain tissue in situ via intracardial perfusion, 2) dissection and removal of whole brain, 3) stationary brain embedding in agarose, 4) precision semi-thick slice preparation using new vibratome instrumentation, 5) clearing brain tissue through a glycerol gradient, and 6) mounting on glass slides for light microscopy and z-stack reconstruction (Figure 1).
For preparing brain slices we implemented a relatively new piece of instrumentation called the 'Compresstome' VF-200 (http://www.precisionary.com/products_vf200.html). This instrument is a semi-automated microtome equipped with a motorized advance and blade vibration system with features similar in function to other vibratomes. Unlike other vibratomes, the tissue to be sliced is mounted in an agarose plug within a stainless steel cylinder. The tissue is extruded at desired thicknesses from the cylinder, and cut by the forward advancing vibrating blade. The agarose plug/cylinder system allows for reproducible tissue mounting, alignment, and precision cutting. In our hands, the 'Compresstome' yields high quality tissue slices for electrophysiology, immunohistochemistry, and direct fixed-tissue mounting and imaging. Combined with optical clearing, here we demonstrate the preparation of semi-thick fixed brain slices for high-resolution fluorescent imaging.

Here we describe a rapid and simple method to image fluorescently labeled cells in semi-thick brain slices. By fixing, slicing, and optically clearing brain tissue we describe how standard epifluorescent or confocal imaging can be used to visualize individual cells and neuronal networks within intact nervous tissue.

A fundamental goal to both basic and clinical neuroscience is to better understand the identities, molecular makeup, and patterns of connectivity that are ...

The overall goal of this procedure is to more easily visualize fluorescent neurons within thick brain slices. This is accomplished by first embedding the brain in an aeros plug holder. The second step of the procedure is to section the brain into thick slices on the compressed tome.The third step of the procedure is to optically clear the brain slices in a glycerol gradient. The final step of the procedure is to mount the cleared brain slices onto slides for fluorescent imaging. Ultimately, results can be obtained that show high resolution fluorescent protein reporter expression in neurons within thick brain slices, through epi fluorescent or confocal microscopy.The main advantage of this technique over existing methods like thin section, serial fluorescent microscopy or multi photon imaging, is that it is much more rapid, less labor intensive and does not require expensive or sophisticated microscopy technique. To begin retrieve the mouse brain tissue that has undergone NC two fixation by cardiac perfusion and has been postfix for one to two hours at four degrees Celsius. Next, make a 2%high strength low gel point agro solution in PBS and heat in the microwave until dissolved.Tran

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세미 두께 뇌 조각의 고해상도 형광 이미징에 대한 빠른 접근

출처: Etienne, F. et al. 급성 뇌 절편에서 ATP 또는 세로토닌에 대한 미세아교세포 과정의 인력에 대한 이광자 이미징. J. Vis. Exp. (2019)이 비디오는 이광자 현미경을 사용하여 뇌 절편에 주입된 화합물에 대한 미세아교세포의 인력을 연구하는 방법을 보여줍니다. 쥐의 뇌 절편을 준비하고, 화학유인제 테스트 화합물을 주입하고, 형광 표지된 미세아교세포를 이미징하여 반응을 시각화하는 방법에 대한 개요를 제공합니다.

쥐 뇌 절편의 화합물에 대한 미세아교세포 돌기 인력을 연구하기 위한 이광자 현미경

출처: Shimojo, H., et al. 쥐 신경 발생 중 노치 신호 전달 역학의 실시간 생물 발광 이미징. J. Vis. 경험. (2019년)이 비디오에서는 형질전환 마우스 배아의 피질 절편 배양을 사용하여 3차원 이미징을 통해 유전자 발현을 모니터링합니다. 생물발광, 형광 및 명시야 이미지는 천연 조직 내에서 신경 분화 동안 실시간 유전자 발현을 시각화하는 데 사용됩니다.

3차원 이미징을 통한 뇌 절편의 실시간 유전자 발현 모니터링

제시 여기 경작 하는 수정 된 롤러 튜브 방법 이며 photoetched coverslips에 정확한 위치와 많은 주 동안 설치류 두뇌의 간헐적인 고해상도 이미징 조각. 신경 생존 및 슬라이스 형태로 잘 유지 됩니다. 셀 형 특정 식에 대 한 바이러스를 사용 하 여이 완전히 동봉 된 시스템의 응용 프로그램에 제공 됩니다.

정확 하 게 지역화 하 고 반복적인 뇌 조각 동봉 된 시스템에서의 장기적인 문화 중 간헐적인 이미징 수정된 롤러 튜브 메서드

Microglia, 두뇌의 거주 면역 세포 그들의 환경의 수정에 형태학 변화 신속 하 게 응답. 이 프로토콜 2 광자 현미경을 사용 하 여 쥐의 급성 뇌 조각에서 세로토닌 또는 ATP microglial 프로세스의 매력을 공부 하는 방법을 설명 합니다.

Microglial 프로세스 매력 ATP 또는 급성 뇌 조각에 세로토닌의 2 광자 영상

전압에 민감한 염료를 사용하여 뇌 조각을 재현하고 안정적으로 광학 기록 하는 방법을 소개합니다. 이 기사에서는 기존의 해마 슬라이스 준비를 사용하여 전압에 민감한 염료 염색 및 광 신호 기록에 대해 설명합니다.

전압에 민감한 염료를 사용하여 뇌 슬라이스의 광자 광자 광기록

여기에서는 축 신장 및 세분화의 맥락에서 마우스 배아의 세 가지 및 네 차원 이미지 데이터를 시각화하고 분석 할 수있는 계산 도구 및 방법을 설명하고, 토토 광학 투영 단층 촬영에서 얻은 다중 광자 현미경을 사용하여 라이브 이미징 및 전체 마운트 면역 형광 염색을 수행합니다.

척추동물 축 신장 및 세분화를 연구하기 위한 세 가지 및 네 차원 시각화 및 분석 접근법

이 프로토콜은 현장 축삭 성장 및 성장 원뿔 역학 을 연구하는 간단하고 강력한 방법을 보여줍니다. 생체외 생리학적으로 관련된 급성 뇌 조각을 준비하는 방법을 설명하고 사용자 친화적인 분석 파이프라인을 제공합니다.

시투에서 Axon Growth and Growth Cone Dynamics in Acute Ex Vivo Embryonic Brain Slice Cultures 시각화

이 프로토콜은 조직 제거 방법인 ScaleSF를 사용하여 뇌 절편에서 신경 세포 영상의 상세한 방법을 제공한다. 이 프로토콜에는 뇌 조직 준비, 조직 정화, 클리어 슬라이스 처리 및 공초점 레이저 스캐닝 현미경 이미징이 포함됩니다.

중시경에서 현미경 비늘에 이르기까지 신경 세포 이미징을위한 조직 제거 방법

유도성 트랜스제닉 계보 추적 마우스 라인을 사용하여 줄기 세포 및 그의 자손을 형광단으로 영구적으로 표시하는 능력은 생체내에서 활성화, 증식, 이동 및/또는 분화의 공간적 및 시간적 분석을 가능하게 한다. 계보 추적은 혈통 헌신, 개입에 대한 반응 및 다능성에 대한 새로운 정보를 나타낼 수 있습니다.

성인 마우스 뇌에서 유도성 형광 표지 줄기 세포의 계보 추적

이 프로토콜은 iDISCO+를 사용하여 온전한 마우스 뇌의 자기 공명 영상, 제거 및 면역 표지를 수행하는 방법을 설명한 다음, 라이트 시트 현미경을 사용한 이미징 및 NuMorph를 사용한 다운스트림 분석에 대한 자세한 설명을 설명합니다.

MRI, 조직 투명화 및 라이트 시트 현미경을 사용한 온전한 신생아 마우스 뇌의 전뇌 단일 세포 이미징 및 분석

본 프로토콜은 (SWITCH - H 2O2 - Antigen Retrieval - 2,2'-thiodiethanol [TDE]) SHORT 조직 형질 전환 기법과 광시트 형광 현미경 이미징을 일상적으로 높은 처리량 프로토콜로 결합하여 수십 개의 사후 인간 뇌 절편의 신속하고 동시적인 광학 투명화, 다중 라운드 라벨링 및 3D 체적 재구성을 위한 단계별 절차를 제공합니다.

Optical Clearing and Labeling for Light-sheet Fluorescence Microscopy in Large-scale Human Brain Imaging(대규모 인간 뇌 영상에서 광시트 형광 현미경을 위한 광학 투명화 및 라벨링)

배우세요 세미 두께 뇌 조각에서 형광 뉴런을 고해상도로 시각화하는 방법을. 뇌 조직을 고정하고 절단한 후 광학적 투명화를 통해 개별 세포와 신경망을 표준 현미경으로 관찰할 수 있습니다.

A Rapid Approach to High-Resolution Fluorescence Imaging in Semi-Thick Brain Slices

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