이 실험실의 안과 연구 작업은 미국의 건강 보조 재단, 진달래 (1972) 기증 기금, 그리고 HKU 소형 프로젝트 기금 (20,097,176,185)에 의해 지원됩니다.

1. 도구 현미경, 니콘 스테레오 탐정, MBF Bioscience - MicroBrightField 주식 회사 2. 준비 <ol><li> 어떤 morphometric 분석 작업을하기 전에 각 망막 샘플은 4 미크론의 두께에 sectioned이며 H &amp; E 염색법를 겪습. </li><li> 상단 주변 지역, 상단 중앙 지역, 낮은 주변 지역, 그리고 낮은 중부 지방 - 망막 부분은 4 지역으로 나누어져 있습니다. </li><li> 영역을 정의 후, 각 지역의 이미지는 니콘의 밝은 빔 현미경 10x40 배율로 취득합니다. </li><li> 이제, 우리는 망막의 morphometry을 분석할 준비가 된 것입니다. </li></ol> 3. INL 두께를 측정 <ol><li> 각 영역의 INL 두께 네 성능은 측정되었다. 포 라인은 INL의 바깥 경계에 윤곽 기준에서 수직 그릴 것입니다. </li><li>스테레오 탐정이라는 소프트웨어를 열고 메인 툴바에서 40x 렌즈를 선택합니다. </li><li> 탭에서 &quot;파일&quot;아래의 &quot;이미지 열기&quot;를 선택합니다. 당신은 분석하고자하는 원하는 망막 지역의 이미지를 선택하십시오. </li><li> 메인 툴바의 드롭 다운 목록 상자에서 윤곽 라인의 유형을 선택하고 &quot;자동 이동&quot;버튼을 누르십시오. </li><li> 오른쪽 추적 창을 클릭하고 &quot;간단한 클릭 추적&quot;를 선택합니다. </li><li> INL의 내부 경계를 따라 등고선 라인을 그립니다. </li><li> 추첨이 완료되면, 오른쪽 추적 창에서 클릭하고 &quot;최종 열기 컨투어&quot;를 선택합니다. </li><li> 마커 도구 모음에서 표시를 선택하고 연결 지점에 마커를 추가할 수 있습니다. </li><li> 모든 마커를 추가한 후, 마커 툴바에있는 마커 아이콘을 취소합니다. </li><li> 마커 중 하나의 중심에 커서를 둡니다. </li></ol><img src="/files/ftp_upload/3377/3377fig1.jpg" alt="그림 1" /><ol start="11"><li> &quot;빨리를 선택하십시오도구 &quot;&quot;탭 아래 &quot;각도를 측정합니다. </li></ol><img src="/files/ftp_upload/3377/3377fig2.jpg" alt="그림 2" /><ol start="12"><li> 오른쪽 추적 창에 클릭하고 &quot;지속&quot;옵션을 해제. </li><li> 첫번째 라인 세그먼트의 끝을 위해 선택한 마커에 인접한 마커의 중심을 클릭하여 각도를 측정합니다. </li></ol><img src="/files/ftp_upload/3377/3377fig3.jpg" alt="그림 3" /><ol start="14"><li> 그런 다음 관련 마커의 중심에 마우스를 이동하고 다시 클릭합니다. </li><li> INL의 바깥 테두리에 고무 밴드 라인을 확장합니다. </li><li> 90 °에 도달할 때까지 각도를 조정합니다. </li></ol><img src="/files/ftp_upload/3377/3377fig4.jpg" alt="그림 4" /><ol start="17"><li> 원하는 위치에 클릭 왼쪽. 각도 측정을 보여주는 대화 상자를 클릭하면 오른쪽에 나타납니다. </li><li> 를 눌러여전히 마우스를 누른 상태에서 &quot;Enter&quot;키를. </li></ol><img src="/files/ftp_upload/3377/3377fig5.jpg" alt="그림 5" /><ol start="19"><li> 줄이 한쪽 끝을에서 새로 선택한 끝에 그려진 있도록 추적 창을 클릭 왼쪽. </li><li> 오른쪽 추적 창을 클릭하고 &quot;최종 열기 컨투어&quot;를 선택합니다. </li></ol><img src="/files/ftp_upload/3377/3377fig6.jpg" alt="그림 6" /><ol start="21"><li> 이전에 설명한대로 정확히 같은 방식으로 다른 위치에 다른 라인을 그립니다. </li><li> 모든 라인을 그리는 후 편집 모드를 선택하고 이에 따라 4 선 세그먼트의 이름을 바꿉니다. </li><li> 네 성능은 &#39;컨투어 측정 &quot;버튼을 누르면 얻을 수있다. </li><li> 이러한 4 가지 측정을 선택하고 &quot;클립 보드에 복사 &#39;를 클릭하면 스프레드 시트로 붙여 넣습니다. </li><li> 선택이 탭에서 &quot;파일&quot;아래의 &quot;다른 이름으로 저장&quot;을 클릭하십시오. 추적 데이터는 데이터 파일로 저장할 수 있습니다. </li></ol><ol><li> 탭에서 &quot;파일&quot;아래의 &quot;이미지 열기&quot;를 선택합니다. 당신은 분석하고자하는 원하는 망막 지역의 이미지를 선택하십시오. </li><li> 메인 툴바의 드롭 다운 목록 상자에서 윤곽 라인의 유형을 선택하십시오. </li><li> 신경 섬유 층 (NFL)의 국경을 따라 등고선 라인을 그립니다. </li><li> 옵션 &quot;최종 열기 컨투어&quot;로 추적을 종료합니다. </li><li> 각 끝에 한 줄의 끝부분에서 마커와 끝 가까이 위치에서 서로를 추가합니다. </li><li> 마커 도구 모음에서 마커 아이콘을 취소합니다. </li><li> 일각에서 마커의 중심에 커서를 둡니다. </li><li> 탭에서 &#39;도구&#39;아래의 &#39;빠른 측정 각도 &quot;를 선택하십시오. </li><li> 오른쪽 추적 창에 클릭하고 &quot;지속&quot;옵션을 해제. </li><li> 고무 밴드 라인을 조정하고 90 °의 각도를 측정합니다. </li><li> 대화 상자가 뜬 후 &quot;Enter&quot;키를 누르십시오. </li><li> 왼쪽 버튼을 클릭 t새로운 라인이 수직 팁에서 확장할 수 있도록 그 창문을 추적. </li><li> 다른 끝에 정확히 같은 방식으로 다른 새로운 라인을 그립니다. </li><li> 두 경계는 RGCs의 수를 계산하는 내에서 만들어집니다. </li><li> 세포 처리가 계산되지 제외 줄에 한 가지 경계를 선택합니다. </li><li> 마커 중 하나 유형을 선택하고 각 라이브 셀 하나를 첨부해 주시기 바랍니다. </li><li> 죽은 세포에 대한 마커의 또 다른 유형을 선택합니다. </li><li> 위치 마커의 각 유형의 숫자​​ 마커 도구 모음에 따라 마커 아이콘과 함께 표시됩니다. </li><li> 편집 모드를 선택하고 윤곽 기준을 선택하십시오. 오른쪽 추적 창에 클릭하고 라인을 바꿉니다. </li><li> 윤곽 기준의 길이는 &quot;컨투어 측정&quot;버튼을 누르면 얻을 수있다. </li><li> 세포의 수는 다음 길이 mm 당 표현할 수있다. </li><li> 선택이 탭에서 &quot;파일&quot;아래의 &quot;다른 이름으로 저장&quot;을 클릭하십시오.레이싱이나 마커 데이터를 데이터 파일로 저장할 수 있습니다. </li></ol> 5. RGC 세포의 세포 크기를 측정 <ol><li> 탭에서 &quot;파일&quot;아래의 &quot;이미지 열기&quot;를 선택합니다. 당신은 분석하고자하는 원하는 망막 지역의 이미지를 선택하십시오. </li><li> 라이브 세포 확대 및 메인 툴바의 드롭 다운 목록 상자에서 윤곽 라인의 유형을 선택합니다. </li><li> 세포의 둘레를 따라 등고선 라인을 그립니다. </li><li> 최종 관한에서는 오른쪽 추적 창을 클릭하고 &quot;최종 가까운 윤곽&quot;를 선택합니다. </li><li> 세포의 영역은 컨투어 측정 대화 상자에 영역의 항목에 표시됩니다. </li></ol>

<ol>
	<li>Lam, T. T., Abler, A. S., Tso, M. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=N-Methyl-D-Aspartate+(NMDA)â&#x20AC;&#x201C;Induced+Apoptosis+in+Rat+Retina.">N-Methyl-D-Aspartate (NMDA)â&#x20AC;&#x201C;Induced Apoptosis in Rat Retina.</a> Investigative Ophthalmology &amp; Visual Science. 40, 2391-2397 (1999).</li><li>Lam, T. T., Abler, A. S., Tso, M. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Apoptosis+and+caspases+after+ischemia-reperfusion+injury+in+rat+retina.">Apoptosis and caspases after ischemia-reperfusion injury in rat retina.</a> Invest. Ophthalmol. Vis. Sci. 40, 967-975 (1999).</li><li>Luo, X. G., Chiu, K., Lau, H. S., Lee, V. W. H., Yung, K. K. L., So, K. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Selective+Vulnerability+of+Retinal+Ganglion+Cells+in+Rat+Chronic+Ocular+Hypertension+Model+at+Early+Phase.">The Selective Vulnerability of Retinal Ganglion Cells in Rat Chronic Ocular Hypertension Model at Early Phase.</a> Cellular and Molecular Neurobiology. 29 (8), 1143-1151 (2009).</li><li>Han, Y. S., Chung, I. Y., Park, J. M., Yu, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuroprotective+effect+of+citicoline+on+retinal+cell+damage+induced+by+kainic+acid+in+rats.">Neuroprotective effect of citicoline on retinal cell damage induced by kainic acid in rats.</a> Korean J. Ophthalmol. 19, 219-226 (2005).</li><li>Hernandez, M., Urcola, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retinal+ganglion+cell+neuroprotection+in+a+rat+model+of+glaucoma+following+brimonidine,+latanoprost+or+combined+treatments.">Retinal ganglion cell neuroprotection in a rat model of glaucoma following brimonidine, latanoprost or combined treatments.</a> Exp. Eye Res. 86, 798-806 (2008).</li><li>Chan, H. C., Chang, R. C. C., Ip, A. K. C., Chiu, K., Yuen, W. H., Zee, S. Y., So, K. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuroprotective+effects+of+Lycium+barbarum+Lynn+on+protecting+retinal+ganglion+cells+in+an+ocular+hypertension+model+of+glaucoma.">Neuroprotective effects of Lycium barbarum Lynn on protecting retinal ganglion cells in an ocular hypertension model of glaucoma.</a> Experimental Neurology. 203, 269-273 (2007).</li></ol>

저자가 공개하는 게 없다.

1. 보다 정확한 두께 측정을 구하는 방법? 이 버튼을 &quot;확대&quot;를 클릭하여 이미지를 확대 및 추적 창에서 클릭두고 있습니다. INL 테두리가 분명하게 볼 수 있습니다. 라인의 끝은 INL의 바깥 경계에 있지 않으면, 우리는 편집 모드에서 라인을 조정할 수 있습니다. 관련 라인을 따기 후 오른쪽 추적 창을 클릭하고 선택 &quot;선택된 윤곽선의 포인트를 넣습니다.&quot; 추가 포인?...

A correction was made to <a href="http://www.jove.com/video/3377">Morphometric Analyses of Retinal Sections</a>. There was an error with an author's name.

Erratum: Morphometric Analyses of Retinal Sections

<table border="1"><tbody><tr><td> 시약의 이름 </td><td> 유형 </td><td> 회사 </td><td> 카탈로그 번호 </td></tr><tr><td> 스테레오 탐정 </td><td> MBF Bioscience 분석 소프트웨어 </td><td> MicroBright 필드 </td><td></td></tr><tr><td> 현미경 </td><td> 올림푸스 BX51 </td><td> 올림푸스 주식 회사 </td><td> BX51 </td></tr></tbody></table>

morphometric analyses of retinal sections

망막 섹션 Morphometric 분석은 망막 질환 검사에 사용되었습니다. 예제의 경우의 연결 세포는 크게 N-메틸-D-aspartate와 쥐 모델에서 망막 신경절 세포 계층 (RGCL)에 분실되었다 (NMDA) excitotoxicity 1, 망막 국소 빈혈 - reperfusion 상해 2, 녹내장 3-유도된. INL 및 내부 plexiform 계층의 감소 (IPL)은 카인산-매개 글루 탐 산염의 excitotoxicity 4로 들어서는 쥐 &#39;눈에는 citicoline 치료 반대했다 두께. RGC 밀도와 소마 크기의 변경은 고가 intraocular 압력 3,5,6과 눈속에서 다른 약물 치료와 함께 관찰되었다. 따라서 망막 morphometries를 분석의 객관적인 방법을 갖는 것은 망막 pathologies 및 치료 전략의 효과를 평가에 큰 의미있을 것으로 생각됩니다. 망막 구조는 멀티 레이어와 뉴런 exi 여러 종류입니다망막에있는 일. 이러한 서로 다른 레이어의 휴대폰 번호, 휴대 크기 및 두께 등 망막의 morphometric 매개 변수는 세포 배양 시스템보다 더 복잡합니다. 초기에 이러한 매개 변수는 다른 상용 이미징 소프트웨어를 사용하여 감지가 가능하다. 값은 상대적인 가치를 일반적이며, 정확한 값으로 변경하면 더 정확한 계산을 할 수 있습니다. 또한, 세포의 크기와 형태의 추적은 특히 만성 녹내장 모델에서 정확하고 통계 분석을위한 충분한 예민하지 않을 수 있습니다. 이 프로토콜에 사용되는 측정은보다 정확하고 편리한 방법을 제공했습니다. 그리고 세포의 라인 및 크기의 절대 길이를 직접 및 기타 파일을 복사할 쉽게 신고 할 수 있습니다. 예를 들어, INL의 안쪽과 바깥쪽 가장 핵의 마진을 추적하고 두께를 측정하는 90도 각도를 그리는 소프트웨어를 사용하여 라인을 형성했다. 소프트웨어의 도움없이는 아마도 경사 라인과 망막 두께의 변화가 있지만개별 관측 사이에서 반복하지. 또한, RGCs의 개수 및 밀도도 계량하실 수 있습니다. 이 프로토콜은 성공적으로 망막의 기능을 quantitating에 다양성을 감소 최소한의 변화를 감지에서 감도를 증가시킵니다. 이 비디오는 망막 섹션 morphometric 분석의 세 가지 유형을 설명합니다. 그들은 INL 두께를 측정 RGCs의 수를 quantifying와 절대값에 RGCs의 크기를 측정 등이 있습니다. 이 세 가지 분석은 스테레오 탐정 (- MicroBrightField 주식 MBF Bioscience)와 함께 진행됩니다. 기술 morphometric 분석을위한 간단하면서도 과학적인 플랫폼을 제공할 수 있습니다.

Watch this Scientific Journal Video about Morphometric Analyses of Retinal Sections at JoVE.com

Morphometric Analyses of Retinal Sections

이 비디오는 망막 신경절 세포의 개수 (RGCs)를 quantifying하고 RGCs의 크기를 측정, 내부 핵 층의 두께를 측정 포함 망막의 morphometric 분석의 세 가지 유형을 보여줍니다. 기술 morphometric 분석을위한 간단하면서도 과학적인 플랫폼을 제공할 수 있습니다.

망막 섹션 Morphometric 분석

Morphometric analyses of retinal sections have been used in examining retinal diseases. For examples, neuronal cells were significantly lost in the ...

morphometric-analyses-of-retinal-sections

Research

JoVE Journal

Neuroscience

10.2K 조회수.  The University of Hong Kong. 이 비디오는 망막 신경절 세포의 개수 (RGCs)를 quantifying하고 RGCs의 크기를 측정, 내부 핵 층의 두께를 측정 포함 망막의 morphometric 분석의 세 가지 유형을 보여줍니다. 기술 morphometric 분석을위한 간단하면서도 과학적인 플랫폼을 제공할 수 있습니다.

비디오: Morphometric Analyses of Retinal Sections

An Isolated Retinal Preparation to Record Light Response from Genetically Labeled Retinal Ganglion Cells

The first steps in vertebrate vision take place when light stimulates the rod and cone photoreceptors of the retina 1. This information is then segregated into what are known as the ON and OFF pathways. The photoreceptors signal light information to the bipolar cells (BCs), which depolarize in response to increases (On BCs) or decreases (Off BCs) in light intensity. This segregation of light information is maintained at the level of the retinal ganglion cells (RGCs), which have dendrites stratifying in either the Off sublamina of the inner plexiform layer (IPL), where they receive direct excitatory input from Off BCs, or stratifying in the On sublamina of the IPL, where they receive direct excitatory input from On BCs. This segregation of information regarding increases or decreases in illumination (the On and Off pathways) is conserved and signaled to the brain in parallel.
The RGCs are the output cells of the retina, and are thus an important cell to study in order to understand how light information is signaled to visual nuclei in the brain. Advances in mouse genetics over recent decades have resulted in a variety of fluorescent reporter mouse lines where specific RGC populations are labeled with a fluorescent protein to allow for identification of RGC subtypes 2 3 4 and specific targeting for electrophysiological recording. Here, we present a method for recording light responses from fluorescently labeled ganglion cells in an intact, isolated retinal preparation. This isolated retinal preparation allows for recordings from RGCs where the dendritic arbor is intact and the inputs across the entire RGC dendritic arbor are preserved. This method is applicable across a variety of ganglion cell subtypes and is amenable to a wide variety of single-cell physiological techniques.

This article provides a description of how to dissect and record from the isolated retinal preparation in mouse. In particular, we describe how to record light responses from a fluorescently labeled ganglion cell population and subsequently identify and analyze its morphology.

유전적으로 망막 신경절 라벨 셀에서 라이트 응답을 기록하기 위해 절연 망막 준비

The first steps in vertebrate vision take place when light stimulates the rod and cone photoreceptors of the retina 1.  This information is then ...

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신경과학

Protocol for Three-dimensional Confocal Morphometric Analysis of Astrocytes

As glial cells in the brain, astrocytes have diverse functional roles in the central nervous system. In the presence of harmful stimuli, astrocytes modify their functional and structural properties, a condition called reactive astrogliosis. Here, a protocol for assessment of the morphological properties of astrocytes is presented. This protocol includes quantification of 12 different parameters i.e. the surface area and volume of the tissue covered by an astrocyte (astrocyte territory), the entire astrocyte including branches, cell body, and nucleus, as well as total length and number of branches, the intensity of fluorescence immunoreactivity of antibodies used for astrocyte detection, and astrocyte density (number/1,000 &#181;m2). For this purpose three-dimensional (3D) confocal microscopic images were created, and 3D image analysis software such as Volocity 6.3 was used for measurements. Rat brain tissue exposed to amyloid beta1-40 (A&#946;1-40) with or without a therapeutic&#160;intervention was used to present the method. This protocol can also be used for 3D morphometric analysis of other cells from either in vivo or in vitro conditions.

Astrocytes in the CNS change their functional and structural properties in response to harmful stimuli. This report presents a protocol for assessment of three-dimensional astrocyte morphology in diseased conditions or after therapeutic interventions.

성상의 3 차원 공 촛점 형태 학적 분석을위한 프로토콜

As glial cells in the brain, astrocytes have diverse functional roles in the central nervous system. In the presence of harmful stimuli, astrocytes modify ...

Immunostaining of Biocytin-filled and Processed Sections for Neurochemical Markers

Electrophysiological recordings of cells using the patch clamp technique have allowed for the identification of different neuronal types based on firing patterns. The inclusion of biocytin/neurobiotin in the recording electrode permits post-hoc recovery of morphological details, which are necessary to determine the dendritic arborization and the regions targeted by the axons of the recorded neurons. However, given the presence of morphologically similar neurons with distinct neurochemical identities and functions, immunohistochemical staining for cell-type-specific proteins is essential to definitively identify neurons. To maintain network connectivity, brain sections for physiological recordings are prepared at a thickness of 300 &#181;m or greater. However, this thickness often hinders immunohistological postprocessing due to issues with antibody penetration, necessitating the resectioning of the tissue. Resectioning of slices is a challenging art, often resulting in the loss of tissue and morphology of the cells from which electrophysiological data was obtained, rendering the data unusable. Since recovery of morphology would limit data loss and guide in the selection of neuronal markers, we have adopted a strategy of recovering cell morphology first, followed by secondary immunostaining. We introduce a practical approach to biocytin filling during physiological recordings and subsequent serial immunostaining for the recovery of morphology, followed by the restaining of sections to determine the neurochemical identity. We report that sections that were filled with biocytin, fixed with paraformaldehyde (PFA), stained, and coverslipped can be removed and restained with a second primary antibody days later. This restaining involves the removal of the coverslip, the washing of sections in a buffer solution, and the incubation of primary and secondary antibodies to reveal the neurochemical identity. The method is advantageous for eliminating data loss due to an inability to recover morphology and for narrowing down the neurochemical markers to be tested based on morphology.

This protocol presents a method for the morphological recovery of neurons patched during electrophysiological recordings using biocytin filling and subsequent immunohistochemical postprocessing. We show that thick biocytin-filled sections that were stained and coverslipped can be restained with a second primary antibody days&#160;or months later.

신경 화학적 마커에 대한 Biocytin 충전 및 가공 섹션의 면역 염색

Electrophysiological recordings of cells using the patch clamp technique have allowed for the identification of different neuronal types based on firing ...

Microstate and Omega Complexity Analyses of the Resting-state Electroencephalography

Microstate and omega complexity are two reference-free electroencephalography (EEG) measures that can represent the temporal and spatial complexities of EEG data and have been widely used to investigate the neural mechanisms in some brain disorders. The goal of this article is to describe the protocol underlying EEG microstate and omega complexity analyses step by step. The main advantage of these two measures is that they could eliminate the reference-dependent problem inherent to traditional spectrum analysis. In addition, microstate analysis makes good use of high time resolution of resting-state EEG, and the four obtained microstate classes could match the corresponding resting-state networks respectively. The omega complexity characterizes the spatial complexity of the whole brain or specific brain regions, which has obvious advantage compared with traditional complexity measures focusing on the signal complexity in a single channel. These two EEG measures could complement each other to investigate the brain complexity from the temporal and spatial domain respectively.

This article describes the protocol underlying electroencephalography (EEG) microstate analysis and omega complexity analysis, which are two reference-free EEG measures and highly valuable to explore the neural mechanisms of brain disorders.

미세 및 휴식 상태 Electroencephalography의 오메가 복잡도 분석

Microstate and omega complexity are two reference-free electroencephalography (EEG) measures that can represent the temporal and spatial complexities of ...

Preparation of Mouse Retinal Cryo-sections for Immunohistochemistry

Preparation of high-quality mouse eye sections for immunohistochemistry (IHC) is critical for assessing the retinal structure and function and for determining the mechanisms underlying retinal diseases. Maintaining structural integrity throughout the tissue preparation is vital for obtaining reproducible retinal IHC data but can be challenging due to the fragility and complexity of retinal cytoarchitecture. Strong fixatives like 10% formalin or Bouin's solution optimally preserve the retinal structure, they often impede IHC analysis by enhancing the background fluorescence and/or diminishing antibody-epitope interactions, a process known as epitope masking. Milder fixatives, on the other hand, like 4% paraformaldehyde, reduces background fluorescence and epitope-masking, meticulous dissection techniques must be utilized to preserve the retinal structure. In this article, we present a comprehensive method to prepare mouse ocular posterior cups for IHC that is sufficient to preserve most antibody-epitope interactions without loss of retinal structural integrity. We include representative IHC with antibodies to various retinal cell type markers to illustrate tissue preservation and orientation under optimal and sub-optimal conditions. Our goal is to optimize IHC studies of the retina by providing a complete protocol from ocular posterior cup dissection to IHC.

This report describes comprehensive methods for preparing frozen mouse retina sections for immunohistochemistry (IHC). Methods described include dissection of the ocular posterior cup, paraformaldehyde fixation, embedding in Optimal Cutting Temperature (OCT) media and tissue orientation, sectioning and immunostaining.

면역 조직 화학을 위한 마우스 망막 극저온 단면도 단면도의 준비

Preparation of high-quality mouse eye sections for immunohistochemistry (IHC) is critical for assessing the retinal structure and function and for ...

Preparation of Newborn Rat Brain Tissue for Ultrastructural Morphometric Analysis of Synaptic Vesicle Distribution at Nerve Terminals

Our laboratory and many others have exploited the high resolving power of transmission electron microscopy to study the morphology and spatial organization of synaptic vesicles. In order to obtain high-quality electron micrographs that can yield the degree of morphological detail necessary for quantitative analysis of pre-synaptic vesicle distribution, optimal specimen preparation is critical. Chemical fixation is the first step in the process of specimen preparation, and of utmost importance to preserve fine ultrastructure. Vascular fixation with a glutaraldehyde-formaldehyde solution, followed by treatment of vibratome-sectioned specimens with osmium tetroxide, stabilizes the maximum number of molecules, especially proteins and lipids, and results in superior conservation of ultrastructure. Tissue is then processed with counterstaining, sequential dehydration and resin-embedding. En bloc staining with uranyl acetate (i.e., staining of vibratome-sectioned tissue before resin embedding) enhances endogenous contrast and stabilizes cell components against extraction during specimen processing. Contrast can be further increased by applying uranyl acetate as a post-stain on ultrathin sections. Double-staining of ultrathin sections with lead citrate after uranyl acetate treatment also improves image resolution, by intensifying electron-opacity of nucleic acid-containing structures through selective binding of lead to uranyl acetate. Transmission electron microscopy is a powerful tool for characterization of the morphological details of synaptic vesicles and quantification of their size and spatial organization in the terminal bouton. However, because it uses fixed tissue, transmission electron microscopy can only provide indirect information regarding living or evolving processes. Therefore, other techniques should be considered when the main objective is to study dynamic or functional aspects of synaptic vesicle trafficking and exocytosis.

We describe procedures for processing newborn rat brain tissue to obtain high-resolution electron micrographs for morphometric analysis of synaptic vesicle distribution at nerve terminals. The micrographs obtained with these methods can also be used to study the morphology of a number of other cellular components and their dimensional structural relationships.

신경 말단에서 시 냅 스 소포 분포의 미세 구조 형태학 적 분석을 위한 신생아 쥐 뇌 조직의 제조

Our laboratory and many others have exploited the high resolving power of transmission electron microscopy to study the morphology and spatial ...

Collection of Frozen Rodent Brain Regions for Downstream Analyses

As our understanding of neurobiology has progressed, molecular analyses are often performed on small brain areas such as the medial prefrontal cortex (mPFC) or nucleus accumbens. The challenge in this work is to dissect the correct area while preserving the microenvironment to be examined. In this paper, we describe a simple, low-cost method using resources readily available in most labs. This method preserves nucleic acid and proteins by keeping the tissue frozen throughout the process. Brains are cut into 0.5&#8211;1.0 mm sections using a brain matrix and arranged on a frozen glass plate. Landmarks within each section are compared to a reference, such as the Allen Mouse Brain Atlas, and regions are dissected using a cold scalpel or biopsy punch. Tissue is then stored at -80 &#176;C until use. Through this process rat and mouse mPFC, nucleus accumbens, dorsal and ventral hippocampus and other regions have been successfully analyzed using qRT-PCR and Western assays. This method is limited to brain regions that can be identified by clear landmarks.

This procedure describes the collection of discrete frozen brain regions to obtain high-quality protein and RNA using inexpensive and commonly available tools.

다운스트림 분석을 위한 냉동 설치류 뇌 영역 모음

As our understanding of neurobiology has progressed, molecular analyses are often performed on small brain areas such as the medial prefrontal cortex ...

Preparation of the Rat Vocal Fold for Neuromuscular Analyses

The purpose of this tutorial is to describe the preparation of the rat vocal fold for histochemical neuromuscular study. This protocol outlines procedures for rat laryngeal dissection, flash-freezing, and cryosectioning of the vocal folds. This study describes how to cryosection vocal folds in both longitudinal and cross-sectional planes. A novelty of this protocol is the laryngeal tracking during cryosectioning that ensures accurate identification of the intrinsic laryngeal muscles and reduces the chance of tissue loss. Figures demonstrate the progressive cryosectioning in both planes. Twenty-nine rat hemi-larynges were cryosectioned and tracked from the emergence of the thyroid cartilage to the appearance of the first section that included the full vocal fold. The full vocal fold was visualized for all animals in both planes. There was high variability in the distance from the appearance of the thyroid cartilage to the appearance of the full vocal fold in both planes. Weight was not correlated to depth of laryngeal landmarks, suggesting individual variability and other factors related to tissue preparation may be responsible for the high variability in the appearance of landmarks during sectioning. This study details a methodology and presents morphological data for preparing the rat vocal fold for histochemical neuromuscular investigation. Due to high individual variability, laryngeal landmarks should be closely tracked during cryosectioning to prevent oversectioning tissue and tissue loss. The use of a consistent methodology, including adequate tissue preparation and awareness of landmarks within the rat larynx, will assist with consistent results across studies and aid new researchers interested in using the rat vocal fold as a model to investigate laryngeal neuromuscular mechanisms.

This protocol describes methods used to prepare rat vocal folds for histochemical neuromuscular study.

신경 근육 분석을위한 쥐 보컬 폴드의 준비

The purpose of this tutorial is to describe the preparation of the rat vocal fold for histochemical neuromuscular study. This protocol outlines procedures ...

Dissection of Single Skeletal Muscle Fibers for Immunofluorescent and Morphometric Analyses of Whole-Mount Neuromuscular Junctions

The neuromuscular junction (NMJ) is a specialized point of contact between the motor nerve and the skeletal muscle. This peripheral synapse exhibits high morphological and functional plasticity. In numerous nervous system disorders, NMJ is an early pathological target resulting in neurotransmission failure, weakness, atrophy, and even in muscle fiber death. Due to its relevance, the possibility to quantitatively assess certain aspects of the relationship between NMJ components can help to understand the processes associated with its assembly/disassembly. The first obstacle when working with muscles is to gain the technical expertise to quickly identify and dissect without damaging their fibers. The second challenge is to utilize high-quality detection methods to obtain NMJ images that can be used to perform quantitative analysis. This article presents a step-by-step protocol for dissecting extensor digitorum longus and soleus muscles from rats. It also explains the use of immunofluorescence to visualize pre and postsynaptic elements of whole-mount NMJs. Results obtained demonstrate that this technique can be used to establish the microscopic anatomy of the synapsis and identify subtle changes in the status of some of its components under physiological or pathological conditions.

The ability to accurately detect neuromuscular junction components is crucial in evaluating modifications in its architecture because of pathological or developmental processes. Here we present a complete description of a straightforward method to obtain high-quality images of whole-mount neuromuscular junctions that can be used to perform quantitative measurements.

면역형광및 전체산 신경근육 접합의 형태 분석에 대한 단일 골격 근육 섬유의 해부

The neuromuscular junction (NMJ) is a specialized point of contact between the motor nerve and the skeletal muscle. This peripheral synapse exhibits high ...

Quantification of Immunostained Caspase-9 in Retinal Tissue

The family of caspases is known to mediate many cellular pathways beyond cell death, including cell differentiation, axonal pathfinding, and proliferation. Since the identification of the family of cell death proteases, there has been a search for tools to identify and expand the function of specific family members in development, health, and disease states. However, many of the currently commercially available caspase tools that are widely used are not specific for the targeted caspase. In this report, we delineate the approach we have used to identify, validate, and target caspase-9 in the nervous system using a novel inhibitor and genetic approaches with immunohistochemical read-outs. Specifically, we used the retinal neuronal tissue as a model to identify and validate the presence and function of caspases. This approach enables the interrogation of cell-type specific apoptotic and non-apoptotic caspase-9 functions and can be applied to other complex tissues and caspases of interest. Understanding the functions of caspases can help to expand current knowledge in cell biology, and can also be advantageous to identify potential therapeutic targets due to their involvement in disease.

Presented here is a detailed immunohistochemistry protocol to identify, validate, and target functionally relevant caspases in complex tissues.