Name: the cutting and floating method for paraffin-embedded tissue for sectioning
Uploaded: 2018-09-05T15:30:00.000+00:00
Duration: 5 min
Description: Watch this Scientific Journal Video about The Cutting and Floating Method for Paraffin-embedded Tissue for Sectioning at JoVE.com

この作品は中国の国家自然科学基金 (許可番号 31400936、31460260) によって支えられた、自然科学財団の江西省の中国 (20171BAB215020)。我々 はまた、この作業を支援する南昌大学とクイーン クイーンメアリー、ロンドン大学との共同プログラムを感謝します。

ここで説明したすべてのメソッドは、動物のケアと使用南昌大学委員会によって承認されています。
1. 機器をアセンブルし、ミクロトームを接続
<ol><li>要件 (補助図 1) あたりパラメーターを設計します。</li>
	<li>アクリル板を製造するローカル工場にパラメーターを送信します。</li>
	<li>シーケンスのすべての部品を組み立てる: クロロホルムを使用してタンクに 7 商業アクリル板を組み合わせてセクション チャネルと水お風呂 (図 1)。 注意: 光と酸素を満たしているとき、クロロホルムは有毒物質を生成します。ヒューム フードの装置を組み立てます。</li>
	<li>アクリル板 #1 (図 2 a,矢印) に穴を介して管状電気加熱要素をインストールします。</li>
	<li>温度コント ローラーをインストールするのには、電気ドリルを使用して #2 アクリル板に小さな穴を作成し、温度検出器 (図 2 a,矢印) をインストールします。タンク (図 2 a) にデジタル サーモスタットをインストールします。 注: 温度検出器は水面下 1 cm 以内にする必要があります。</li>
	<li>電源アクセスを提供するために、機器の電源を入れる前に 24 V 以下の電圧を調整します。</li>
	<li>ミクロトームから切片屑トレーを取り外します。</li>
	<li>中立的なシリコーン ・ シーラント (図 2 b) ミクロトーム ナイフ ホルダーを含むセクション チャネルにリンクします。 注: 刃の上の上の中立シリコーン ・ シーラント中傷されず古い刃は簡単に交換できるようにします。</li>
	<li>クロロホルムと固化する中立的なシリコン シール剤の時間を許可するために一晩乾燥させます。</li>
	<li>ブレード ホルダーに刃をインストールします。(図 2) 風呂の水に水を注ぐ。水表面だけブレード (図 2 D,アロー ・ ヘッド) の上部に触れる必要があります。</li>
</ol>2. パラフィン切片
注: 破片は、多くの場合ワックスのブロックと水の表面の上下端に蓄積します。この残骸は、定期的にクリアする必要があります。
<ol><li>
従来のパラフィン切片10を実行します。
	<ol>
<li>回転式ミクロトームのパラフィン包埋標本をクランプし、ブレード ホルダーにブレードを配置します。</li>
		<li>ハンド ホイールを切り、標本をセクションします。</li>
		<li>パラフィン切片のリボンを 38.5 ° C の温水浴に転送します。</li>
		<li>顕微鏡にセクションを展開した後の水面からスライドをピックアップします。</li>
		<li>42 ° C のオーブンでスライドを一晩乾かします。</li>
	</ol>
</li>
	<li>
改良されたパラフィン切片 (図 3)
	<ol>
<li>機器の電源を。</li>
		<li>温度検出スイッチでヒーターを開き、40.0 ° C 1 時間事前に水を暖めるために 38.0 ° C 間ターゲットの温度を設定します。</li>
		<li>パラフィン包埋にしたパラフィン ブロックを変更します。ミクロトームのカセット クランプにパラフィン ブロックを配置し、それをロックします。 注意: パラフィン ブロックの優れたと劣るボーダーは、円弧を形成し、狭いセクション チャネルを通過することができないからセクション リボンを防ぐために水平方向に変更ください。</li>
		<li>切断より厚いセクションでサンプルの迅速なアプローチします。</li>
		<li>適切なセクションの厚さ (成体マウスの脳、成体マウス腎臓、胎児マウスの脳に 10 μ m、ゼブラフィッシュの目 4 μ m) を調整します。ハンド ホイールを切り、セクションは、セクション チャネルと風呂に自動的に入力されます。</li>
		<li>ゆっくりと均一な切断ストローク (図 3 a) で標本をセクションします。 注: ハンド ホイールが数分間停止した後の最初のセクションが変更されます。セクションは連続した傷がある場合、替刃を置き換える必要があります。</li>
		<li>ライン (図 3 b、3 C) で浮かぶ風呂室にセクションをガイドします。 注: 場合は head セクションは、チャンバー壁に準拠して、浮いた状態を保つにブラシ ガイドを使用します。</li>
		<li>顕微鏡スライド上にセクションを遵守します。セクションの後完全に風呂の水、ピンセットで別のいくつかのセクションで展開します。顕微鏡のスライド (図 3 D) に風呂の水からそれらをピックアップします。 注: セクションする必要がありますピックアップする水のバスの後方からセクション チャネルと水お風呂の前のそれらがまだできないので完全に展開。</li>
		<li>一晩 42 ° C のオーブンでそれらを配置することによって、スライドを乾燥します。スライドは、無期限に室温で格納されることができます。</li>
	</ol>
</li>
	<li>ヘマトキシリン ・ エオジン (HE) 染色法の改良8の評価のために使用します。</li>
</ol>

<ol>
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著者は、このデバイスの特許を持っているし、競争の興味を宣言しません。

パラフィン セクション形態を改善し、従来のパラフィン切片中の無駄な時間の問題を解決するには、改良されたパラフィン切片作製法切削と展開を組み合わせたを作成しました。この改良法は、セクション チャネル、水浴と温度検出スイッチとヒーターで構成されるシンプルな機器に依存します。セクション リボン セクション チャネルを介して水の風呂に入るし、自動的に切削しながら?...

形態学的研究は、生物学の研究が重要です。新技術は、全体の組織または生物1,2,3、薄い部分に試料を切断から直接彼らの目標は続いて染色を観察する研究者を許可しているが、プライマリのまま法者は唯一組織形態も組織に直接ターゲット蛋白質。光学顕微鏡を使用して、3 つのセクション タイプ: パラフィン、冷凍、およびグリコールメタクリル。セクショニングは共通組織抗原性を保護するため、試料の準備は簡単です、保有組織形態が悪いと薄い断面4,5には不向き。パラフィン切片は、保存状態の良い形態を展示するため最もよく使用される方法です。標本は完全に脱水し、ワックスに埋め込まれた、パラフィン ブロックに無期限に格納できます。また、パラフィン切片さらに実験の生物学的プローブ アクセスを改善し、Z 方向に細胞層オーバーレイを削減する薄いセクションを生成します。
しかし、従来のパラフィン切片がかかり、オペレーターのスキルを要求します。パラフィン切片を固定、脱水、埋め込み、切り取り、およびフローティング受けます。重要なは、風呂の水にナイフ ホルダーからセクション リボンを転送するは、ジュニアのオペレーターのためには必要なは難しいです。乾いた空気で特にセクション リボン静電気によるねじれが、暖かい水面に展開しにくい。セクションを改善するために品質、氷の水でそれらを浸すことまたはミクロトーム近く加湿器で湿度を上げるワックス ブロックを冷却のミクロトーム刃パス間公開される組織表面を潤し、6、7をお勧めします.ハイブリッド パラフィン包埋、セクショニング8、および商業部門転送システム支援9パラフィン切片を改善するための新しいメソッドが含まれます。これらのメソッドは、部分的にパラフィンを向上させる彼らははるかに厄介な断面を作る、商務部搬送システムは高価なセクショニング速度と品質。
このプロトコルでは、簡単、安価で柔軟な装置ステップバイ ステップ、回転式ミクロトームのナイフ ホルダーに接続することができますを作成する方法を示します。この装置は、温度検出スイッチ付きのヒーター、水風呂セクション チャネルで構成されます。切断した後、セクションの数十はセクション チャネルに流れるし、風呂の水、直接このように自動的に展開を入力します。これはパラフィン切片の効率が向上し、この技術をより便利になります。このメソッドよりアダルト マウス海馬セクション、成体マウス腎臓セクション、胚 15.5 日齢 (E15.5) マウスの脳のセクション、およびセクションを短い時間で収穫し、形態学的によりそのまま残った大人ゼブラフィッシュ眼を使用します。このメソッドは、加速されたパラフィン セクション区別の喪失を回避しながら断面を必要とする他の組織サンプルの使用もできます。

<table><tbody><tr><td>Incubator</td><td>Boekel Scientific</td><td>133000-2</td><td></td></tr><tr><td>Ethanol&amp;nbsp;</td><td>Sinopharm Chemical Reagent Co.,Lid</td><td>64-17-5</td><td></td></tr><tr><td>Xylene&amp;nbsp;</td><td>Sinopharm Chemical Reagent Co.,Lid</td><td>1330-20-7</td><td></td></tr><tr><td>Paraplast</td><td>Leica</td><td>39601006</td><td></td></tr><tr><td>Heated Paraffin Embedding Module</td><td>Leica</td><td></td><td></td></tr><tr><td>Commercial acrylic board</td><td></td><td></td><td></td></tr><tr><td>Trichloromethane</td><td>Sinopharm Chemical Reagent Co.,Lid</td><td>67-66-3</td><td></td></tr><tr><td>Tubular electric heating element(12 V 200 W)</td><td></td><td></td><td></td></tr><tr><td>Temperature controller(12 V 120 W)</td><td>Mingsuo</td><td>XH-W3002</td><td></td></tr><tr><td>Rotary microtome&amp;nbsp;</td><td>Leica</td><td></td><td></td></tr><tr><td>Neutral silicone sealant</td><td></td><td></td><td>Link the water channel with the microtome knife holder</td></tr><tr><td>Voltage transformer</td><td>Dearll</td><td>S-250-12</td><td></td></tr><tr><td>Disposable blade</td><td>Accu-Edge</td><td>4689</td><td></td></tr><tr><td>Hematoxylin</td><td>Baso Diagnostics Inc.</td><td>BA-4025</td><td></td></tr><tr><td>Eosin&amp;nbsp;</td><td>Baso Diagnostics Inc.</td><td>BA-4025</td><td></td></tr><tr><td>Microslide</td><td>Sail Brand</td><td>7105</td><td></td></tr><tr><td>Neutral balsam</td><td>Sinopharm Chemical Reagent Co.,Lid</td><td>10004160</td><td></td></tr><tr><td>Coverslip&amp;nbsp;</td><td>Citoglas</td><td>10212424C</td><td></td></tr><tr><td>Microscope</td><td>Carl Zeiss</td><td></td><td></td></tr><tr><td>Hydrochloric acid</td><td>Xilong Chemical</td><td>7647-01-0</td><td></td></tr><tr><td>Water bath for paraffin sections</td><td>Leica</td><td></td><td></td></tr><tr><td>HistoCore Arcadia C&amp;nbsp;- Cold Plate</td><td>Leica</td><td></td><td></td></tr><tr><td>paraffin repellent spray&amp;nbsp;</td><td>Thermo Scientific</td><td>9990420</td><td></td></tr></tbody></table>

the cutting and floating method for paraffin-embedded tissue for sectioning

組織学や病理学のパラフィン包埋組織の断面が使用されています。ただし、それは面倒です。この方法を改善するためにいくつかの民間企業は、液体の水を使用して複雑な部分転送システムを考案しています。この技術を簡素化し、我々 はカットとシンプルな恒温室; 内フローティングを組み合わせた自家製の機器を使用して単純なメソッドを作成したがって、セクションは自動的に水表面における水の風呂を入力します。アダルト ゼブラフィッシュ目マウス萌芽期の脳、成体マウス腎臓成体マウス脳海馬は、比較のため従来のパラフィン切片と提案手法の両方を使用して切られました。統計分析は、我々 の改良法時間を保存したし、高い品質のセクションを示しています。さらに、短時間で全体の標本のパラフィン切片ジュニア演算子の簡単です。

改善方法は、そのままパラフィン セクションの数を高めた。成体マウス海馬組織、成体マウス腎臓、マウス萌芽期の脳、ゼブラフィッシュの目のこの新しいメソッドをテストしました。タンクに水を追加し、40.0 ° c. に 38.0 ° C 水の温度に維持されました。準備した後、順次組織サンプル、切断され、従来区分と比較しています。新しいメソッドは、断面欠損を回避?...

Watch this Scientific Journal Video about The Cutting and Floating Method for Paraffin-embedded Tissue for Sectioning at JoVE.com

The Cutting and Floating Method for Paraffin-embedded Tissue for Sectioning

切削とフローティング区分のための組織のパラフィン包埋法

ここでは、パラフィン切片を改善するためのプロトコルを提案する.このメソッドは、切断と従来の方法に必要な転送処理を避けるために簡単な恒温室を使用して浮動小数点を兼ね備えています。その結果、そのままパラフィン切片数と効率が大幅に向上。

Sectioning of the paraffin-embedded tissue is widely used in histology and pathology. However, it is tedious. To improve this method, several commercial ...

the-cutting-floating-method-for-paraffin-embedded-tissue-for

Research

JoVE Journal

Neuroscience

18.2K ビュー.  Nanchang University. ここでは、パラフィン切片を改善するためのプロトコルを提案する.このメソッドは、切断と従来の方法に必要な転送処理を避けるために簡単な恒温室を使用して浮動小数点を兼ね備えています。その結果、そのままパラフィン切片数と効率が大幅に向上。

ビデオ: The Cutting and Floating Method for Paraffin-embedded Tissue for Sectioning

Thin Sectioning of Slice Preparations for Immunohistochemistry

Many investigations in neuroscience, as well as other disciplines, involve studying small, yet macroscopic pieces or sections of tissue that have been preserved, freshly removed, or excised but kept viable, as in slice preparations of brain tissue.  Subsequent microscopic studies of this material can be challenging, as the tissue samples may be difficult to handle.  Demonstrated here is a method for obtaining thin cryostat sections of tissue with a thickness that may range from 0.2-5.0 mm.  We routinely cut 400 micron thick Vibratome brain slices serially into 5-10 micron coronal cryostat sections.  The slices are typically first used for electrophysiology experiments and then require microscopic analysis of the cytoarchitecture of the region from which the recordings were observed.  We have constructed a simple device that allows controlled and reproducible preparation and positioning of the tissue slice.  This device consists of a cylinder 5 cm in length with a diameter of 1.2 cm, which serves as a freezing stage for the slice.  A ring snugly slides over the cylinder providing walls around the slice allowing the tissue to be immersed in freezing compound (e.g., OCT).  This is then quickly frozen with crushed dry ice and the resulting  wafer  can be position easily for cryostat sectioning.  Thin sections can be thaw-mounted onto coated slides to allow further studies to be performed, such as various staining methods, in situ hybridization, or immunohistochemistry, as demonstrated here.

The present method allows reproducible cryostat sectioning of small, difficult-to-manage, tissue pieces, such as biopsies and brain slices. We utilize a simple aluminum freezing stage to facilitate handling of tissue and a standard cryostat to routinely produce 5-10 micron serial sections from 400 micron thick brain slices.

免疫組織化学のためのスライス標本の薄いセクショニング

Many investigations in neuroscience, as well as other disciplines, involve studying small, yet macroscopic pieces or sections of tissue that have been ...

生物学

Sectioning Mammary Gland Whole Mounts for Lesion Identification

Normal mammary gland development may be altered by exposure to environmental toxicants and pharmaceutical products, excessive exposure to hormones, and genetic alterations. Mammary gland whole mounts are an inexpensive method to capture the progression of morphological changes that may arise after exposure. However, in later life, when abnormalities are more prone to develop, sole reliance on this one method may not always provide enough information to make a proper diagnosis of the abnormality. Historically, in chemical test guideline studies, a single mammary gland is removed at necropsy and prepared as a hematoxylin and eosin (H&#38;E)-stained section. The incorporation of contralateral mammary whole-mount collection and analysis decreases the likelihood of a false-negative assessment. Evaluation of the whole mount is limited by the presence of one or two entire mammary glands on a slide, and in some cases, the abnormalities observed in the whole mount are not uniformly represented in the H&#38;E section.&#160; The goal of this study was to develop a protocol for converting coverslipped mammary whole mounts to H&#38;E-stained sections so that lesions that would otherwise have been missed or that are difficult to diagnose can be identified. Here, we detail a method to produce a high-quality, paraffin-embedded H&#38;E section from a mammary gland that was initially prepared as a whole mount. In comparison to a tissue that was intentionally prepared for H&#38;E sectioning, the whole mount requires additional preparation for tissue removal and processing. However, this method is considered inexpensive, as it requires common lab reagents and little additional time. As a result, this method can provide invaluable information on how chemical and environmental exposures alter normal mammary development, as well as display changes that occur because of genetic modifications.

We developed a method to successfully remove, process, section, and stain, for histopathological evaluation, mammary tissue that had originally been fixed on slides as whole mounts. This method may promote the collection and evaluation of mammary gland whole mounts in reproductive and developmental test guideline studies.

病変同定のための乳腺全体マウントの分割

Normal mammary gland development may be altered by exposure to environmental toxicants and pharmaceutical products, excessive exposure to hormones, and ...

がん研究

Paraffin Embedding and Thin Sectioning of Microbial Colony Biofilms for Microscopic Analysis

Sectioning via paraffin embedding is a broadly established technique in eukaryotic systems. Here we provide a method for the fixation, embedding, and sectioning of intact microbial colony biofilms using perfused paraffin wax. To adapt this method for use on colony biofilms, we developed techniques for maintaining each sample on its growth substrate and laminating it with an agar overlayer, and added lysine to the fixative solution. These optimizations improve sample retention and preservation of micromorphological features. Samples prepared in this manner are amenable to thin sectioning and imaging by light, fluorescence, and transmission electron microscopy. We have applied this technique to colony biofilms of Pseudomonas aeruginosa, Pseudomonas synxantha, Bacillus subtilis, and Vibrio cholerae. The high level of detail visible in samples generated by this method, combined with reporter strain engineering or the use of specific dyes, can provide exciting insights into the physiology and development of microbial communities.

We describe fixation, paraffin embedding, and thin sectioning techniques for microbial colony biofilms. In prepared samples, biofilm substructure and reporter expression patterns can be visualized by microscopy.

パラフィン包埋と顕微解析の微生物コロニー バイオ フィルムの薄い断面

Sectioning via paraffin embedding is a broadly established technique in eukaryotic systems. Here we provide a method for the fixation, embedding, and ...

免疫・感染学

Preparation of Non-human Primate Brain Tissue for Pre-embedding Immunohistochemistry and Electron Microscopy

Despite all the technological advances at the light microscopy&#160;level, electron microscopy remains the only tool in neuroscience to examine and characterize ultrastructural and morphological details of neurons, such as synaptic contacts. Good preservation of brain tissue for electron microscopy can be obtained by rigorous cryo-fixation methods, but these techniques are rather costly and limit the use of immunolabeling, which is crucial to understand the connectivity of identified neuronal systems. Freeze-substitution methods have been developed to allow the combination of cryo-fixation with immunolabeling. However, the reproducibility of these methodological approaches usually relies on costly freezing devices. Moreover, achieving reliable results with this technique is very time-consuming and skill-challenging. Hence, the traditional chemically fixed brain, particularly with acrolein fixative, remains a time-efficient and low-cost method to combine electron microscopy with immunohistochemistry. Here, we provide a reliable experimental protocol using chemical acrolein fixation that leads to the preservation of primate brain tissue and is compatible with pre-embedding immunohistochemistry and transmission electron microscopic examination.

Here, we provide an easy, low-cost, and time-efficient protocol to chemically fix primate brain tissue with acrolein fixative, allowing for long-term preservation that is compatible with pre-embedding immunohistochemistry for transmission electron microscopy.

プレ埋め込み免疫組織化学および電子顕微鏡のための非ヒト霊長類脳組織の調製

Despite all the technological advances at the light microscopy&#160;level, electron microscopy remains the only tool in neuroscience to examine and ...

神経科学

Establishment and Analysis of Tumor Slice Explants As a Prerequisite for Diagnostic Testing

Organotypic primary tissue explant cultures, which include precision-cut slices, represent the three-dimensional (3-D) tissue architecture as well as the multicellular interactions of native tissue. Tissue&#160;slices immediately cut from freshly resected tumors preserve spatial aspects of intratumor heterogeneity (ITH), thus making them useful surrogates of in vivo biology. Careful optimization of tissue slice preparation and cultivation conditions is fundamental for the predictive diagnostic potential of tumor slice explants. In this regard, murine models are valuable, as these provide a consistent flow of tumor material to perform replicate and reproducible experiments. This protocol describes the culturing of murine lung tumor-derived tissue slices using a rotating incubation unit, a system that enables intermittent exposure of tissues to oxygen and nutrients. Our previous work showed that the use of rotating incubation units improves the viability of tissue compared to other culture methods, particularly floating slices and stagnant filter supports. Here, we further show that slice thickness influences the viability of cultured slices, suggesting that optimization of slice thickness should be done for different tissue types. Pronounced ITH in relevant oncogenic functions, such as signaling activities, stromal cell infiltration or expression of differentiation markers, necessitates evaluation of adjacent tissue slices for the expression of markers altered by drug treatment or cultivation itself. In summary, this protocol describes the generation of murine lung tumor slices and their culture on a rotating incubation unit and demonstrates how slices should be systematically analyzed for the expression of heterogeneous tissue markers, as a prerequisite prior to drug response studies.

We provide a method for the generation, cultivation and systematic analysis of organotypic slices derived from murine lung tumors. We also describe how to optimize for slice thickness, and how to select drug concentrations to treat tumor slices.

診断テストのための前提条件として外植体腫瘍スライスの樹立と解析

Organotypic primary tissue explant cultures, which include precision-cut slices, represent the three-dimensional (3-D) tissue architecture as well as the ...

Hybrid-Cut: An Improved Sectioning Method for Recalcitrant Plant Tissue Samples

Maintaining plant section integrity is essential for studying detailed anatomical structures at the cellular, tissue, or even organ level. However, some plant cells have rigid cell walls, tough fibers and crystals (calcium oxalate, silica, etc.), and high water content that often disrupt tissue integrity during plant tissue sectioning.
This study establishes a simple Hybrid-Cut tissue sectioning method. This protocol modifies a paraffin-based sectioning technique and improves the integrity of tissue sections from different plants. Plant tissues were embedded in paraffin before sectioning in a cryostat at -16 &#176;C. Sectioning under low temperature hardened the paraffin blocks, reduced tearing and scratching, and improved tissue integrity significantly. This protocol was successfully applied to calcium oxalate-rich Phalaenopsis orchid tissues as well as recalcitrant tissues such as reproductive organs and leaves of rice, maize, and wheat. In addition, the high quality of tissue sections from Hybrid-Cut could be used in combination with in situ hybridization (ISH) to provide spatial expression patterns of genes of interest. In conclusion, this protocol is particularly useful for recalcitrant plant tissue containing high crystal or silica content. Good quality tissue sections enable morphological and other biological studies.

This protocol describes a simple Hybrid-Cut tissue sectioning method that is useful for recalcitrant plant tissues. Good quality tissue sections enable anatomical studies and other biological studies including in situ hybridization (ISH).

ハイブリッドカット：手に負えない植物組織試料のための改善されたセクショニング方法

Maintaining plant section integrity is essential for studying detailed anatomical structures at the cellular, tissue, or even organ level. However, some ...

Pancreatic Islet Embedding for Paraffin Sections

Experiments using isolated pancreatic islets are important for diabetes research, but islets are expensive and of limited abundance. Islets contain a mixed cell population in a structured architecture that impacts function, and human islets are widely variable in cell type composition. Current frequently used methods to study cultured islets include molecular studies performed on whole islets, lumping disparate islet cell types together, or microscopy or molecular studies on dispersed islet cells, disrupting islet architecture. For in vivo islet studies, paraffin-embedded pancreas sectioning is a powerful technique to assess cell-specific outcomes in the native pancreatic environment. Studying post-culture islets by paraffin sectioning would offer several advantages: detection of multiple outcomes on the same islets (potentially even the exact-same islets, using serial sections), cell-type-specific measurements, and maintaining native islet cell-cell and cell-substratum interactions both during experimental exposure and for analysis. However, existing techniques for embedding isolated islets post-culture are inefficient, time consuming, prone to loss of material, and generally produce sections with inadequate islet numbers to be useful for quantifying outcomes. Clinical pathology laboratory cell block preparation facilities are inaccessible and impractical for basic research laboratories. We have developed an improved, simplified bench-top method that generates sections with robust yield and distribution of islets. Fixed islets are resuspended in warm histological agarose gel and pipetted into a flat disc on a standard glass slide, such that the islets are distributed in a plane. After standard dehydration and embedding, multiple (10+) 4 - 5 &#181;m sections can be cut from the same islet block. Using this method, histological&#160;and immunofluorescent analyses can be performed on mouse, rat, and human islets. This is an effective, inexpensive, time-saving approach to assess cell-type-specific, intact-architecture outcomes from cultured islets.

Ex vivo pancreatic islet studies are important for diabetes research. Existing techniques to study cultured islets in their native 3-dimensional architecture are time consuming, inefficient, and infrequently used. This work describes a new, simple, and efficient method for generating high-quality paraffin sections of whole cultured islets.

パラフィン セクションの埋め込み膵島

Experiments using isolated pancreatic islets are important for diabetes research, but islets are expensive and of limited abundance. Islets contain a ...

Microscopy Techniques for Interpreting Fungal Colonization in Mycoheterotrophic Plants Tissues and Symbiotic Germination of Seeds

Structural botany is an indispensable perspective to fully understand the ecology, physiology, development, and evolution of plants. When researching mycoheterotrophic plants (i.e., plants that obtain carbon from fungi), remarkable aspects of their structural adaptations, the patterns of tissue colonization by fungi, and the morphoanatomy of subterranean organs can enlighten their developmental strategies and their relationships with hyphae, the source of nutrients. Another important role of symbiotic fungi is related to the germination of orchid seeds; all Orchidaceae species are mycoheterotrophic during germination and seedling stage (initial mycoheterotrophy), even the ones that photosynthesize in adult stages. Due to the lack of nutritional reserves in orchid seeds, fungal symbionts are essential to provide substrates and enable germination. Analyzing germination stages by structural perspectives can also answer important questions regarding the fungi interaction with the seeds. Different imaging techniques can be applied to unveil fungi endophytes in plant tissues, as are proposed in this article. Freehand and thin sections of plant organs can be stained and then observed using light microscopy. A fluorochrome conjugated to wheat germ agglutinin can be applied to the fungi and co-incubated with Calcofluor White to highlight plant cell walls in confocal microscopy. In addition, the methodologies of scanning and transmission electron microscopy are detailed for mycoheterotrophic orchids, and the possibilities of applying such protocols in related plants is explored. Symbiotic germination of orchid seeds (i.e., in the presence of mycorrhizal fungi) is described in the protocol in detail, along with possibilities of preparing the structures obtained from different stages of germination for analyses with light, confocal, and electron microscopy.

This protocol aims to provide detailed procedures for collecting, fixing, and maintaining mycoheterotrophic plant samples, applying different microscopy techniques such as scanning and transmission electron microscopy, light, confocal, and fluorescence microscopy to study fungal colonization in plants tissues and seeds germinated with mycorrhizal fungi.

菌従属栄養植物組織における真菌のコロニー形成と種子の共生発芽を解釈するための顕微鏡技術

Structural botany is an indispensable perspective to fully understand the ecology, physiology, development, and evolution of plants. When researching ...

Optimized Thin Endometrium Mouse Model with Low Mortality and Minimal Adhesions

Establishing a Mouse Model of Thin Endometrium

Thin endometrium (TE) has been widely recognized as a critical cause of infertility. However, the pathogenesis of TE remains unclear, and satisfactory treatment options are still urgently needed. Several animal models of TE have been developed, but the mouse model involving abdominal surgery and injection of 95% ethanol presents a formidable challenge due to the high mortality rate and risk of intrauterine adhesions if not performed correctly. Here, we describe a detailed protocol that produces reliable and reproducible TE with a very low mortality rate and minimal intrauterine adhesions by injecting 95% ethanol into the mouse uterus with varying infusion times. The results showed that all of the mice successfully developed TE with infusion times ranging from 1-3 min, characterized by a typical reduction in endometrial thickness and the number of glands, as well as excessive endometrial fibrosis. These findings suggest that this mouse model is suitable for studying thin endometrium and can serve as a platform for developing future TE treatments.

This article describes a detailed protocol for producing a reliable and reproducible thin endometrium with a very low mortality rate and minimal intrauterine adhesions by injecting 95% ethanol into the mouse uterus within 1-3 min.

Thin endometrium (TE) has been widely recognized as a critical cause of infertility. However, the pathogenesis of TE remains unclear, and satisfactory ...

Formalin-Fixed Paraffin Embedded (FFPE) Tissue Preservation: A Method for Studying Tissue Morphology

Source: Nagaraj A.S., et al., <a href="https://www.jove.com/v/58569/establishment-analysis-tumor-slice-explants-as-prerequisite-for">Establishment and Analysis of Tumor Slice Explants As a Prerequisite for Diagnostic Testing.</a> J. Vis. Exp. (2018)
The following video describes how to preserve morphology of a tissue with formalin fixation and paraffin embedding.

切削とフローティング区分のための組織のパラフィン包埋法

要約

さらに動画を探す

この動画の章

免疫組織化学のためのスライス標本の薄いセクショニング

病変同定のための乳腺全体マウントの分割

パラフィン包埋と顕微解析の微生物コロニーバイオフィルムの薄い断面

プレ埋め込み免疫組織化学および電子顕微鏡のための非ヒト霊長類脳組織の調製

診断テストのための前提条件として外植体腫瘍スライスの樹立と解析

ハイブリッドカット：手に負えない植物組織試料のための改善されたセクショニング方法

パラフィンセクションの埋め込み膵島

菌従属栄養植物組織における真菌のコロニー形成と種子の共生発芽を解釈するための顕微鏡技術

Establishing a Mouse Model of Thin Endometrium

ホルマリン固定パラフィン包埋(FFPE)組織保存:組織形態の研究方法

切削とフローティング区分のための組織のパラフィン包埋法

要約

さらに動画を探す

この動画の章

免疫組織化学のためのスライス標本の薄いセクショニング

病変同定のための乳腺全体マウントの分割

パラフィン包埋と顕微解析の微生物コロニー バイオ フィルムの薄い断面

プレ埋め込み免疫組織化学および電子顕微鏡のための非ヒト霊長類脳組織の調製

診断テストのための前提条件として外植体腫瘍スライスの樹立と解析

ハイブリッドカット：手に負えない植物組織試料のための改善されたセクショニング方法

パラフィン セクションの埋め込み膵島

菌従属栄養植物組織における真菌のコロニー形成と種子の共生発芽を解釈するための顕微鏡技術

Establishing a Mouse Model of Thin Endometrium

ホルマリン固定パラフィン包埋(FFPE)組織保存:組織形態の研究方法

パラフィン包埋と顕微解析の微生物コロニーバイオフィルムの薄い断面

パラフィンセクションの埋め込み膵島