Name: do's and don'ts in the preparation of muscle cryosections for histological analysis
Uploaded: 2015-05-15T13:00:00.000+00:00
Duration: 10 min 27 s
Description: Watch this Scientific Journal Video about Do's and Don'ts in the Preparation of Muscle Cryosections for Histological Analysis at JoVE.com

We would like to thank Alex Miller for manuscript revisions. This work was supported with the research grants awarded to M.G. from Cure CMD, Struggle Against Muscular Dystrophy (SAM) and Muscular Dystrophy Association (218938).

1. Doku Hasat ve kas Dokuların Donma <ol><li> Izofluran (2-kloro-2- (diflorometoksi) -1,1,1-trifloro-etan) aşırı dozda fare öldürülür. Davlumbaz bu adımı gerçekleştirin ve böyle bir kurutucuda veya izofluran batırılmış bir pamuklu çubuk içeren bir fanus gibi bir ikincil çevreleme yöntemini kullanın. </li><li> Sıkıca Kapkaççı sıkarak ölümü onaylayın. Böyle servikal dislokasyon olarak ötenazi ikincil yöntemi kullanın. </li><li> Kaslar gevşek saç tellerinin yapışmasını azaltmak için% 70 alkol ile kürk ıslatın. Böyle 5. olarak ince bir forseps kullanarak uzuv kapalı cilt soyma. </li><li> Dikkatle, altından kemiklerden (bu durumda tibialis anterior (TA)) ilgi kas ayrı tendonu başlayarak çıkarın. </li><li> , Kas tüketim OCT (Optimum Kesme Sıcaklığı bileşik) onu karşılamak ve etiketli atılabilir dondurma kalıp üzerinde düz yatıyordu. Kas tai için normal fizyolojik oryantasyon (başında olduğundan emin olungermeden l). Alternatif olarak, küçük strafor karelere kas dondurmak ve kasların doğru uzunluğunu sağlamak için böcek işaretçilerine kullanarak pin. </li><li> Sıvı azot kullanılarak çelik beherglasta 2-metilbütan (izopentan) soğutun. 5 dakika - Bu ortalama 3 alır. Beyaz çökeltiler beher alt ve duvarlarında oluşturmak başlayana kadar aralıklı karıştırarak tutun. NOT: Bu durumda, izopentan uygun donma sıcaklığına ulaşmıştır (-150 ° C) </li><li> Soğutulmuş 2-metilbütana kasları içeren kalıpları batırın. 20 sn - 15 gastroknemius soleus (GS) ve triceps gibi 12 sn ve daha büyük kaslar - Böyle diyafram ve ekstansör digitorum longus 6 (EDL) gibi küçük kasları dondurun. Çok uzun bu çatlama neden olabileceğinden için kasları donma yok. </li><li> Kuru buz üzerinde donmuş dokuları aktarın ve izopentan buharlaşması (bu işlem etrafında ortalama 15 alır unutmayın - 20 dakika) izin verin. Alüminyum folyo dokuları sarın ve u saklayınlökotrien antagonistlerinin düşük sıcaklık kesit kadar (-70 ° C ila -80 ° C). </li></ol> 2. Doku Blok Hazırlanır OCT Dokuları katıştırma <ol><li> Kuru buz üzerinde Taşımacılık dokuları çözülme önlemek için cyrostat için. Doku transferi ve bunları, 30 dakika için dengelenmeye bırakın (doku tam çözülme önlemek için -20 -24 ° C&#39;ye ayarlanmış) bölme kriostada. </li><li> Peltier defne soğutma açın. Kriyostat bu soğutma yoksa, hızlı OCT dondurmak için aerosol soğutma sprey kullanın. Bu donma hasara yol açabilir olarak gömme sırasında herhangi bir noktada kas çözülme izin vermeyin. </li><li> Örnek diskte Ekim düzgün bir ince katman ekleyin ve soğumaya bırakın. OCT en durumdayken sıvı üst alt donmuş zaman, (boyuna kesitler için OCT enine kesitler ve paralel için OCT dik) doğru yönde de doku yerleştirin. Hızlı OCT dondurmak için aerosol soğutma sprey kullanın. Isı ext ile doku unembeded bölümünü dokununreaktöre verilmiş ve 45 saniye bekleyin. </li><li> , Kas çevresinde Ekim başka ince bir tabaka ekleyin aerosol soğutma sprey ile sprey ve bir önceki adımda olduğu gibi ısı ayıklayın. </li><li> Bütün kas kaplıdır kadar sprey ve ısı çıkarıcı soğutma aerosol bir arada kullanarak Ekim dondurmak hızla küçük artışlarla Ekim ekleyerek tutun. </li><li> Doku bloğun üstünde ısı çıkarıcı koyun ve 5 dakika bekleyin. </li></ol> 3. Bölüm hazırlanması ve Kasların Orta göbek Bölgesi&#39;nden Bölümler belirlenmesi <ol><li> Numune kafasına örnek monte edin. Diski sabitlemek için topuzu sıkın. Numune disk numune kafası ile iyi temas halinde olduğundan emin olun. </li><li> ° -21 ila -24 ° C&#39;ye odası sıcaklığını ayarlamak ve ayarlanan sıcaklık elde edilene kadar bekleyin. </li><li> Kalınlık ayarlarını (7 mikron) ayarlayın; ilerleyen ya da kaba ve ince ayarlarını kullanarak bloğu geri çekilmeden tarafından blok kırpın. </li><li> Bölümleri toplayınüzerinde slayt kesme bölümlerinin yapışmasını kolaylaştırmak için zıt ücretleri arasındaki cazibe kullanarak bölümleri üzerine slayt üst basarak pozitif yüklü mikroskop slaytlar önceden ısıtılmış. </li><li> Bölümlerin enine kesit alanı (CSA) ölçerek orta göbek bölümleri tanımlamak. Mikroskop bağlı olduğu bilgisayarda bu kullanarak görüntüleme yazılımı yapın. NOT: Faz kontrastlı görüntüler alınır ve tüm kas çevresi yazılım programına bir izleme fonksiyonu üzerinden ölçülür. Bu, her iki CSA ve asgari gelincik çapı ölçümleri (bir tanecik karşıt taraflarında iki paralel teğet arasındaki en küçük mesafe olarak ifade edilir lif enine kesit boyutunun ölçü) ürün elde edilir. Not: CSA ve minimum gelincik çap ölçümleri plato başladığınızda, orta göbek ulaşıldı. </li><li> Envanter gelecekteki boyama -80 ° C&#39;de slaytlar arşivlemek ve. NOT: lekelenme için Ekim öncesinde kaldırılması gerekmezg işlemleri. Slaytlar kaplandı sonra, hemen boyanabilir hazırız. </li></ol> 4. Boyama NOT: Ayrıntılı adım adım prosedürleri ürün veri sayfasında bulunabilir ve boyama işlemleri için takip edilmesi gerekirken, kişisel optimizasyon optimum boyama elde etmek için gerekli olabilir. <ol><li> Ortam (tabloya bakınız), montaj, su ile karışmayan olduğu için, (% 70,% 95 ve% 100 etanol içinde 2 dk) alkol artan sınıflar üzerinde kaydığı dihidrat ve ksilen (5 dakika için% 100) sağlamak için bunları temizlemek slaytlar zamanla opak olmazlar. </li><li> Bölümleri lamel ve kurumaya bırakın. Mikroskop ile görüntü görüntüleme yazılımı ile donatılmış. </li></ol>

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The authors have nothing to disclose.

Histoloji ve immünohistokimya çeşitli nöromüsküler bozukluklar tezahürü ve patoloji ilerlemesini anlamada anahtar bir araç olarak kullanılmıştır. Klasik kas biyopsileri ilk formaldehit sabit ve daha sonra parafin mumu ile gömüldü. Formaldehit tespit mimari dokusu koruyan ve oda sıcaklığında doku depolama ve taşıma izin verirken, aynı zamanda doğru immün elde etmek için daha ileri adımlar gerektiren enzimler ve çapraz bağlarının proteinleri inaktive eder. Bu sorun donmuş kesitlerin kullan?...

Kas histoloji incelenmesi nöromüsküler bozuklukların 1-4 farklı anlamada kritik bir rol oynamaktadır. Diğer tekniklerle birlikte, bu araştırmacıların patoloji tezahürü ve ilerlemesinin daha iyi bir fikir almak için ve aynı zamanda bir terapötik müdahale 5-10 etkinliğini değerlendirmek için gerekli olabilir izin verir. Eksizyon hemen önce fizyolojik durumunu korumak amacıyla Ancak, doğru olması için, dokular uygun şekilde korunması gerekir. Bu kaldırma, koruma, kesit ve boyama esnasında büyük dikkat gerektirir. Dokular, ışık mikroskobu çalışma için iki farklı yolla hazırlanabilirler. (FFPE) bölümleri, gömülü ilk yöntem, formalin sabit parafin dokularının parafin mumu 11,12 ile gömülü önce% 10 doğal tamponlu formaldehit sabit gerektirir. tespit aşaması aynı zamanda çapraz bağlantılar endojen pr daha iyi morfolojisi korumak için yardımcı oluroteins böylece immün için karmaşık antijen alma prosedürleri gerektiren ve bu bölümlerde 12 enzimatik boyama olasılığını ortadan kaldırır. Dondurulmuş kesitler, diğer taraftan, önceden sabit veya işlenmesine gerek yoktur; Bu nedenle, proteinler doğal biyolojik konformasyonlar 13,14 muhafaza edebiliyoruz. Bundan başka, dondurulmuş kesitler de bazen sarkomlar ve diğer cerrahi biyopsi 15, ameliyat tanısında, örneğin gerekli olan hızlı gerçekleştirme süresi, izin verir. Düzgün yapılmazsa Ancak, bu yöntem histolojik inceleme için bölümler elverişsiz hale kas mimarisi değişiklikleri tanıtır zararı, don eğilimli. Bu kağıt düzgün muayene için en uygun boyama elde etmek için kas biyopsi hazırlamak için en etkili yöntem açıklayacağım.

<table><tbody><tr><td>Cryostat</td><td>Leica CM 1850</td><td>Leica Microsystems Inc.</td><td>Buffalo Grove, IL 60089 U.S.A., office phone 1-800-248-0123&lt;br /&gt;Alternative: Richard Allen Scientific</td></tr><tr><td>Microscope</td><td>Nikon Eclipse 50i</td><td>Nikon Instruments Inc.</td><td>1300 Walt Whitman Road, Melville, N.Y. 11747-3064 U.S.A., Phone: 1-631-547-8500&lt;br /&gt;Alternative: Olympus</td></tr><tr><td>Image analysis software</td><td>Nikon Imaging Software Basic Research</td><td>Nikon Instruments Inc.</td><td>1300 Walt Whitman Road, Melville, N.Y. 11747-3064, U.S.A., Phone: 1-631-547-8500&lt;br /&gt;Alternatives: Image J, Metamorph</td></tr><tr><td>Isoflurane</td><td>14043-220-05</td><td>JD Medical Dist. Co., Inc.</td><td>1923 West Peoria Avenue, Phoenix, AZ 85029 U.S.A.</td></tr><tr><td>OCT</td><td>4583</td><td>Sakura Inc.</td><td>Torrence, CA 90501 U.S.A.&lt;br /&gt;Alternative source: Leica Microsystem</td></tr><tr><td>Freeze It</td><td>23-022524</td><td>Thermofisher</td><td>790 Memorial Dr, Cambridge, MA, 02139 U.S.A.&lt;br /&gt;Alternative source: VWR Scientific</td></tr><tr><td>Disposable tissue mold</td><td>22-363-554</td><td>Thermofisher</td><td>790 Memorial Dr, Cambridge, MA 02139 U.S.A.&lt;br /&gt;Alternative source: VWR Scientific</td></tr><tr><td>Colorfrost plus slides</td><td>9991001</td><td>Thermofisher</td><td>790 Memorial Dr, Cambridge, MA 02139 U.S.A.&lt;br /&gt;Alternative source: VWR Scientific</td></tr><tr><td>Acetone</td><td>650501-4L</td><td>Sigma-Aldrich</td><td>3050 Spruce Street, St. Louis, MO 63103 U.S.A.&lt;br /&gt;Alternative source: VWR Scientific</td></tr><tr><td>Ethyl alcohol</td><td>HC-1300-1GL</td><td>Thermofisher</td><td>790 Memorial Dr, Cambridge, MA 02139 U.S.A.&lt;br /&gt;Alternative source: VWR Scientific</td></tr><tr><td>2-methyl butane</td><td>M 32631-SL</td><td>Sigma-Aldrich</td><td>3050 Spruce Street, St. Louis, MO 63103 U.S.A.&lt;br /&gt;Alternative source: VWR Scientific</td></tr><tr><td>Xylene</td><td>HC-7001GA</td><td>Thermofisher</td><td>790 Memorial Dr, Cambridge, MA 02139 U.S.A.&lt;br /&gt;Alternative source: VWR Scientific</td></tr><tr><td>Cytoseal 280</td><td>8311-4</td><td>Thermofisher</td><td>790 Memorial Dr, Cambridge, MA 02139 U.S.A.&lt;br /&gt;Alternative source: VWR Scientific</td></tr><tr><td>Hematoxylin Gill #3</td><td>CS402-1D</td><td>Thermofisher</td><td>790 Memorial Dr, Cambridge, MA 02139 U.S.A.&lt;br /&gt;Alternative source: VWR Scientific</td></tr><tr><td>Eosin</td><td>6766007</td><td>Thermofisher</td><td>790 Memorial Dr, Cambridge, MA 02139 U.S.A.&lt;br /&gt;Alternative source: VWR Scientific</td></tr></tbody></table>

do's and don'ts in the preparation of muscle cryosections for histological analysis

Kas biyopsi histolojik değerlendirilmesi nöromüsküler bozuklukların patoloji gelişimi ve ilerlemesinde anlamada vazgeçilmez bir araç olarak hizmet vermiştir. Ancak, bunu yapmak için, uygun bakım eksize ve optimum boyama elde etmek için dokular korunması sırasında alınması gerekir. Doku koruma yöntemlerinden biri parafin mumu ile gömerek sonra formaldehit dokuları tamir ve kapsar. Bu yöntem de morfoloji koruyan ve oda sıcaklığında uzun süreli depolama için izin verir, ancak hantal ve toksik kimyasalların kullanımını gerektirir. Bundan başka, etkili bir immün için antijen alma protokolleri gerektiren antijeni çapraz bağlayabilen, formaldehit tespit sonuçları. Aksine, dondurulmuş kesit tespiti gerektiren ve bu nedenle, biyolojik antijen yapısını muhafaza etmez. Bu yöntem aynı zamanda intraopera gibi hızlı histolojik değerlendirme gerektiren durumlarda özellikle yararlıdır hale sefer hızlı sırayla ayrı bir avantaj sağlarCerrahi biyopsi tive. Burada farklı histolojik ve immünolojik lekeleri ile görselleştirme için kas biyopsisi hazırlamak en etkili yöntem tarif.

hasat ve dokuların dondurulması için set-up Şekil 1&#39;de görülebilir. Kesilen dokular kurutma (Şekil 1A) önlemek için PBS batırılmış bir gazlı bez üzerine saklanmalıdır. Dokular da doğru morfolojisi (Şekil 1B) sağlamak için Ekim batırılmış ve cryo kalıp düzenlendiği doğru yönde ve mümkün olduğunca fizyolojik uzunluğuna yakın olmalıdır histolojik analiz için kullanılmak üzere seçildiğinde. Bir doku dondurma için müsait izopen...

Watch this Scientific Journal Video about Do's and Don'ts in the Preparation of Muscle Cryosections for Histological Analysis at JoVE.com

Do's and Don'ts in the Preparation of Muscle Cryosections for Histological Analysis

Here we demonstrate the most efficient methods for freezing, embedding, cryosectioning, and staining of muscle biopsies to avoid freezing artifacts.

Histolojik Analiz kas cryosections Hazırlanmasında Yapılması ve Yapılmaması Gerekenler

Histological evaluation of muscle biopsies has served as an indispensable tool in the understanding of the development and progression of pathology of ...

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31.8K Görüntüleme.  Boston University. Here we demonstrate the most efficient methods for freezing, embedding, cryosectioning, and staining of muscle biopsies to avoid freezing artifacts.

Video: Do's and Don'ts in the Preparation of Muscle Cryosections for Histological Analysis

Induction of Acute Skeletal Muscle Regeneration by Cardiotoxin Injection

Skeletal muscle regeneration is a physiological process that occurs in adult skeletal muscles in response to injury or disease. Acute injury-induced skeletal muscle regeneration is a widely used, powerful model system to study the events involved in muscle regeneration as well as the mechanisms and different players. Indeed, a detailed knowledge of this process is essential for a better understanding of the pathological conditions that lead to skeletal muscle degeneration, and it aids in identifying new targeted therapeutic strategies. The present work describes a detailed and reproducible protocol to induce acute skeletal muscle regeneration in mice through a single intramuscular injection of cardiotoxin (CTX). CTX belongs to the family of snake venom toxins and causes myolysis of myofibers, which eventually triggers the regeneration events. The dynamics of skeletal muscle regeneration is evaluated by histological analysis of muscle sections. The protocol also illustrates the experimental procedures for dissecting, freezing, and cutting the Tibialis Anterior muscle, as well as the routine Hematoxylin &#38; Eosin staining that is widely used for subsequent morphological and morphometric analysis.

This manuscript describes a detailed protocol to induce acute skeletal muscle regeneration in adult mice and subsequent manipulations of the muscles, such as dissection, freezing, cutting, routine staining, and myofiber cross-sectional area analysis.

Cardiotoxin Enjeksiyonu ile Akut İskelet Kası Regeneration indüksiyon

Skeletal muscle regeneration is a physiological process that occurs in adult skeletal muscles in response to injury or disease. Acute injury-induced ...

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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.

Nöromüsküler Analizler için Sıçan Vokal Kıvrımının Hazırlanması

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

Nörobilim

Tissue Triage and Freezing for Models of Skeletal Muscle Disease

Skeletal muscle is a unique tissue because of its structure and function, which requires specific protocols for tissue collection to obtain optimal results from functional, cellular, molecular, and pathological evaluations. Due to the subtlety of some pathological abnormalities seen in congenital muscle disorders and the potential for fixation to interfere with the recognition of these features, pathological evaluation of frozen muscle is preferable to fixed muscle when evaluating skeletal muscle for congenital muscle disease. Additionally, the potential to produce severe freezing artifacts in muscle requires specific precautions when freezing skeletal muscle for histological examination that are not commonly used when freezing other tissues. This manuscript describes a protocol for rapid freezing of skeletal muscle using isopentane (2-methylbutane) cooled with liquid nitrogen to preserve optimal skeletal muscle morphology. This procedure is also effective for freezing tissue intended for genetic or protein expression studies. Furthermore, we have integrated our freezing protocol into a broader procedure that also describes preferred methods for the short term triage of tissue for (1) single fiber functional studies and (2) myoblast cell culture, with a focus on the minimum effort necessary to collect tissue and transport it to specialized research or reference labs to complete these studies. Overall, this manuscript provides an outline of how fresh tissue can be effectively distributed for a variety of phenotypic studies and thereby provides standard operating procedures (SOPs) for pathological studies related to congenital muscle disease.

The analysis of skeletal muscle tissues to determine structural, functional, and biochemical properties is greatly facilitated by appropriate preparation. This protocol describes appropriate methods to prepare skeletal muscle tissue for a broad range of phenotyping studies.

Doku Triyaj ve İskelet Kası Hastalıkları Modeller için Donma

Skeletal muscle is a unique tissue because of its structure and function, which requires specific protocols for tissue collection to obtain optimal ...

Biyoloji

Cryosectioning Method for Microdissection of Murine Colonic Mucosa

The colonic mucosal tissue provides a vital barrier to luminal antigens. This barrier is composed of a monolayer of simple columnar epithelial cells. The colonic epithelium is dynamically turned over and epithelial cells are generated in the stem cell containing crypts of Lieberk&#252;hn. Progenitor cells produced in the crypt-bases migrate toward the luminal surface, undergoing a process of cellular differentiation before being shed into the gut lumen. In order to study these processes at the molecular level, we have developed a simple method for the microdissection of two spatially distinct regions of the colonic mucosa; the proliferative crypt zone, and the differentiated surface epithelial cells. Our objective is to isolate specific crypt and surface epithelial cell populations from mouse colonic mucosa for the isolation of RNA and protein.

Here we describe a simple method for the isolation of spatially distinct murine colonic epithelial surface cells from the underlying crypt-base cells.

Fare Kolon Mukozanın Mikrodisseksiyon için Cryosectioning Yöntemi

The colonic mucosal tissue provides a vital barrier to luminal antigens. This barrier is composed of a monolayer of simple columnar epithelial cells. The ...

Fiber Type and Subcellular-Specific Analysis of Lipid Droplet Content in Skeletal Muscle

Skeletal muscle lipid infiltration, known as myosteatosis, increases with obesity and ageing. Myosteatosis has also recently been discovered as a negative prognostic factor for several other disorders such as cardiovascular disease and cancer. Excessive lipid infiltration decreases muscle mass and strength. It also results in lipotoxicity and insulin resistance depending on total intramyocellular lipid content, lipid droplet (LD) morphology, and subcellular distribution. Fiber type (oxidative vs glycolytic) is also important, since oxidative fibers have a greater capacity to utilize lipids. Because of their crucial implications in pathophysiology, in-depth studies on LD dynamics and function in a fiber type-specific manner are warranted.
Herein, a complete protocol is presented for the quantification of intramyocellular lipid content and analysis of LD morphology and subcellular distribution in a fiber type-specific manner. To this end, serial muscle cryosections were stained with the fluorescent dye Bodipy and antibodies against myosin heavy chain isoforms. This protocol enables the simultaneous processing of different muscles, saving time and avoiding possible artifacts and, thanks to a personalized macro created in Fiji, the automatization of LD analysis is also possible.

Increasing evidence indicates that excessive infiltration of lipids inside skeletal muscle results in lipotoxicity and diabetes. Here, we present a complete protocol, including tissue processing, staining with Bodipy, image acquisition, and analysis, to quantify the size, density, and subcellular distribution of lipid droplets in a fiber-type specific manner.

İskelet Kasında Lipid Damlacık İçeriğinin Lif Tipi ve Subhücresel-Spesifik Analizi

Skeletal muscle lipid infiltration, known as myosteatosis, increases with obesity and ageing. Myosteatosis has also recently been discovered as a negative ...

Tıp

High-Throughput, Multi-Image Cryohistology 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.

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.

Yüksek Verim, Mineralize Dokuların Çoklu Resim Cryohistology

There is an increasing need for efficient phenotyping and histopathology of a variety of tissues. This phenotyping need is evident with the ambitious ...

Cryosectioning of Contiguous Regions of a Single Mouse Skeletal Muscle for Gene Expression and Histological Analyses

With this method, consecutive cryosections are collected to enable both microscopy applications for tissue histology and enrichment of RNA for gene expression using adjacent regions from a single mouse skeletal muscle. Typically, it is challenging to achieve adequate homogenization of small skeletal muscle samples because buffer volumes may be too low for efficient grinding applications, yet without sufficient mechanical disruption, the dense tissue architecture of muscle limits penetration of buffer reagents, ultimately causing low RNA yield. By following the protocol reported here, 30 &#956;m sections are collected and pooled allowing cryosectioning and subsequent needle homogenization to mechanically disrupt the muscle, increasing the surface area exposed for buffer penetration. The primary limitations of the technique are that it requires a cryostat, and it is relatively low throughput. However, high-quality RNA can be obtained from small samples of pooled muscle cryosections, making this method accessible for many different skeletal muscles and other tissues. Furthermore, this technique enables matched analyses (e.g., tissue histopathology and gene expression) from adjacent regions of a single skeletal muscle so that measurements can be directly compared across applications to reduce experimental uncertainty and to reduce replicative animal experiments necessary to source a small tissue for multiple applications.

Consecutive cryo-sections are collected to enable histological applications and enrichment of RNA for gene expression measurements using adjacent regions from a single mouse skeletal muscle. High-quality RNA is obtained from 20 - 30 mg of pooled cryosections and measurements are directly compared across applications.

Gen İfade ve histolojik Analizler Tek Fare İskelet Kası bitişik Bölgeler Cryosectioning

With this method, consecutive cryosections are collected to enable both microscopy applications for tissue histology and enrichment of RNA for gene ...

A Multi-hole Cryovial Eliminates Freezing Artifacts when Muscle Tissues are Directly Immersed in Liquid Nitrogen

Studies on skeletal muscle physiology face the technical challenge of appropriately processing the specimens to obtain sections with clearly visible cytoplasmic compartments. Another hurdle is the tight apposition of myofibers to the surrounding tissues. Because the process of tissue fixation and paraffin embedding leads to the shrinkage of muscle fibers, freezing is an optimal means of hardening muscle tissue for sectioning. However, a commonly encountered issue, the formation of ice crystals, occurs during the preparation of frozen sections because of the high water content of muscle. The protocol presented here first describes a simple and efficient method for properly freezing muscle tissues by immersing them in liquid nitrogen. The problem with using liquid nitrogen alone is that it causes the formation of a nitrogen gas barrier next to the tissue, which acts as an insulator and inhibits the cooling of the tissues. To avoid this "vapor blanket" effect, a new cryovial was designed to increase the speed of liquid flow around the tissue surface. This was achieved by punching a total of 14 inlet holes in the wall of the vial. According to bubble dynamics, a higher rate of liquid flow results in smaller bubbles and fewer chances to form a gas barrier. When liquid nitrogen flows into the cryovial through the inlet holes, the flow velocity around the tissue is fast enough to eliminate the gas barrier. Compared to the method of freezing muscle tissues using pre-chilled isopentane, this protocol is simpler and more efficient and can be used to freeze muscle in a throughput manner. Furthermore, this method is optimal for institutions that do not have access to isopentane, which is extremely flammable at room temperature.

This protocol describes a procedure to freeze muscle tissues by plunging them directly into liquid nitrogen. This protocol also highlights a new cryovial that can avoid the &#34;blanket effect&#34; of nitrogen gas when liquid nitrogen contacts the tissue surface of a specimen.

Kas Dokular doğrudan sıvı azot içine girdikleri zaman Artifacts Donma bir çoklu-delikli dondurarak saklamaya uygun vialler ortadan kaldırır

Studies on skeletal muscle physiology face the technical challenge of appropriately processing the specimens to obtain sections with clearly visible ...

In Situ Immunofluorescent Staining of Autophagy in Muscle Stem Cells

Increasing evidence points to autophagy as a crucial regulatory process to preserve tissue homeostasis. It is known that autophagy is involved in skeletal muscle development and regeneration, and the autophagic process has been described in several muscular pathologies and age-related muscle disorders. A recently described block of the autophagic process that correlates with the functional exhaustion of satellite cells during muscle repair supports the notion that active autophagy is coupled with productive muscle regeneration. These data uncover the crucial role of autophagy in satellite cell activation during muscle regeneration in both normal and pathological conditions, such as muscular dystrophies. Here, we provide a protocol to monitor the autophagic process in the adult Muscle Stem Cell (MuSC) compartment during muscle regenerative conditions. This protocol describes the setup methodology to perform in situ immunofluorescence imaging of LC3, an autophagy marker, and MyoD, a myogenic lineage marker, in muscle tissue sections from control and injured mice. The methodology reported allows for monitoring the autophagic process in one specific cell compartment, the MuSC compartment, which plays a central role in orchestrating muscle regeneration.

In Situ Immunofluorescent Staining of Autophagy in Muscle Stem Cells

Active autophagy is associated with productive muscle regeneration, which is essential for Muscle Stem Cell (MuSC) activation. Here, we provide a protocol for the in situ detection of LC3, an autophagy marker in MyoD-positive MuSCs of muscle tissue sections from control and injured mice.

Otofajinin Kas Hücrelerinde Yerinde İmmunofloresan Boyanması

Increasing evidence points to autophagy as a crucial regulatory process to preserve tissue homeostasis. It is known that autophagy is involved in skeletal ...

Immunolabelling Myofiber Degeneration in Muscle Biopsies

The necrosis of muscle fibres (myonecrosis) plays a central role in the pathogenesis of several muscle conditions, including muscular dystrophies. Therapeutic options addressing the causes of muscular dystrophy pathogenesis are expected to alleviate muscle degeneration. Therefore, a method to assay and quantify the extent of cell death in muscle biopsies is needed. Conventional methods to observe myofiber degeneration in situ are either poorly quantitative or rely on the injection of vital dyes. In this article, an immunofluorescence protocol is described that stains necrotic myofibers by targeting immunoglobulin G (IgG) uptake by myofibers. The IgG uptake method is based on cell features characterizing the necrotic demise, including 1) the loss of plasma membrane integrity with the release of damage-associated molecular patterns and 2) the uptake of plasmatic proteins. In murine cross-sections, the co-immunolabelling of myofibers, extracellular matrix proteins, and mouse IgG allows clean and straightforward identification of myofibers with necrotic fate. This simple method is suitable for quantitative analysis and applicable to all species, including human samples, and does not require the injection of vital dye. The staining of necrotic myofibers by IgG uptake can also be paired with other co-immunolabelling.

Described here is a protocol for direct immunolabelling of necrotic myofibers in muscle cryosections. Necrotic cells are permeable to serum proteins, including immunoglobulin G (IgG). Revealing the uptake of IgG by myofibers allows the identification and quantification of myofibers that undergo necrosis regardless of muscle condition.