著者は、彼女の研究室の専門知識のための樹脂の埋め込みとMsステファニの墓の中に彼女の専門知識にMSスーコネルの支援に感謝したい。著者は、彼らのアドバイスを教授フランクKandzioraと博士マリー=アンヌPolbothに感謝したいと思います。

<ol><li>組織は、10％のリン酸塩を含む密閉容器にホルマリン溶液3をバッファに配置されます。蛍光分析は蛍光色素が光に敏感であるため、実行する場合は、光に最小限の露出を確認してください。 </li><li>組織の方向が要求される確立。 </li><li>サイズの試料をトリミング、正しい向きで、バンドを使用して見た。安全対策が守られることを確認します。試料はその後不透明な容器に入れている、の量は十分な固定を実現するために試料の約10倍のサイズにする必要があります。試料は、ホルマリン溶液で、そのサイズに応じて1から2週間の合計ままにしてください。それは、テストと最適化のために制御の組織を用いて提案されている。 </li><li>組織は、埋め込み型にフィットし、70％エタノールを含む密閉不透明な容器にティッシュを置く確認してください。 </li><li>軽いからと一定の撹拌下シールド、エタノールの濃度を昇順で組織を処理する。 <ol class="lalpha"><li> 70％エタノール1週間</li><li> 80％エタノール1週間</li><li> 90％エタノール1週間</li><li> 100％エタノール1週間</li></ol></li><li>組織はその後、再び光から、一定の撹拌下シールド、1週間ブタノールにクリアされます。 </li><li> 樹脂と骨の密度を厳密に一致する必要がある。樹脂の密度が変更（ステップ8を参照）、我々は適切な密度を確保する最初の試験片を埋め込 ​​むことをお勧めすることができます。我々は、光学顕微鏡と蛍光色素分析のためにTechnovit 7100（ヘレウスKulzer）を使用してください。免疫組織化学が必要な場合は代替の樹脂は、そのようなTechnovit 9100（ヘレウスKulzer）として使用する必要があります。 </li><li> Technovit 7100調製液は、必要に応じて、キットに付属の軟化ソリューションの小アリコートと混合してもよいが、せいぜい5％に調製液の量は、我々の経験で軟化ソリューションはほとんど必要ありません。無水エタノールとベースの液体Technovit 7100等しい部分は光の露出を最小限に抑え、24時間のプレ浸透液とし、撹拌しながら混合し、組織に適用されます。 </li><li>浸透溶液を100mlのベース液に溶解さ1gの硬化剤I（= 1袋）を使用して構成されています。これは、事前に浸透液を置換し、一定の撹拌下に1週間のために潜入するために許可されています。 </li><li>切削面を下向きにして、金型に組織を置きます。樹脂のための組織の周囲の余白が（理想的には最低5ミリ）があることを確認、これは、ブロックの強度を確保。硬化剤II 1mlでTechnovit 7100調製液の15mlsを混合し、金型の試料の周りに注ぐと、少なくとも5 millimietresでそれをカバー。硬化時間は2時間以内に開始されますが、一晩完全になります。 </li><li>取付けは、次にTechnovit 3040（ヘレウスKulzer）を使用してバッキングブロックをfashioningによって実行されます。粉1部の液体の粘稠な液体を得るために2部の体積比でTechnovit 3040を混在させる。ブロックのベース上に2 mm以上のレベルまでブロックの背面にあるくぼみにTechnovit 3040を注ぐ。約10分後に金型を剥がし、ブロックの準備が完了することができます。 </li><li>密度、したがって、メタクリル樹脂の切削特性は、部屋の周囲の湿度の影響を受けやすくなっています。ブロックは、したがって、デシケーター中に保管してください。 </li><li>地上のセクショニングは厚さ20から50マイクロメートルの間に大きなセクションを生成します。厚いセクションでは、明るい蛍光を生成するとして、それは（下のセクションを参照）蛍光色素の分析に便利です。 macrotomeにダイヤモンドの刃を固定します。 macrotome上の貯水池に、石油スピリットなどの潤滑剤を、追加。クランプでブロックを固定し、ブレードに親しませる。十分な潤滑を確保する（セクションあたり約20-30分）をゆっくりと発生する研削が可能です。最初のセクションでは、切断面を公開し、セクションが、それは破棄されなければorientates。 </li><li>次のセクションは、Superfrostプラススラ​​イド上に配置されます。セクションでは、我々はその上にサンドイッチ用の袋の部分を配置し、別のスライド位置にクランプを持つそれは平らな保有を推奨するようカールする傾向を持っています。その後、1時間摂氏60から80度の間のインキュベータに入れられます。これは、メタクリル酸を柔らかくし、セクションが平坦な方法で、スライドに付着することができます。 </li><li>穏やかな方法で、細かいグリットのサンドペーパーを使用して、必要に応じて地上部を洗浄し、研磨されることがあります。 </li><li>そりのミクロトームは、光microsocopyに最適なセクションを提供する薄いセクションを、カットします。それはこれらのハードIPブロックを切断するために必要な剛性を、追加したとして、そりのミクロトームを使用してください。またパワーロータリーミクロトームを使用することができます。シャープなステンレススチールの刃を使用してください。これは、1つが使用されている他のようにシャープにすることができるように利用可能な2つのブレードを有することが好ましい。刃は、ブロックで45度の角度を確認する必要があります。蘇rfaceの軟化は、必要に応じてブロックするために湿紙を適用することによって達成することができます。それは質のセクションを取得するために必要な切削条件を達成するためにいくつかのセクションがかかる場合があります。セクションは、水浴に上場し、上記16のようにスライド上に配置されます。 </li><li>染色試薬は、以下の資料に記載されています。プロトコルは、バンクロフト4らに基づいています。厚さのばらつきに起因するそれは、テストスライドの汚れを最適化することをお勧めします。ラックの染色は、組織が必要とされるフラットスライドに直接汚れを追加するようにスライドをオフフロートする可能性があります。同一のプロトコルまたはラックの染色は、組織形態計測に必要な一貫性のある染色成果のために必要です。 <ol class="lalpha"><li>ヘマトキシリン​​とエオシン<ol class="lroman"><li> Haematoxyilin 5〜10分で染色</li><li>スコットの水道水でよく洗う</li><li>エオシン5分で染色</li><li>水道水で洗う</li><li>キシレンでクリア</li><li>マウント</li><li> 結果 <ol><li>類骨=ピンク</li><li>石灰化骨=紫褐色</li><li>核=青</li></ol></li></ol></li><li>フォンコッサ<ol class="lroman"><li>硝酸銀溶液の場所と石灰化骨が黒くなるまで、強い光にさらす（約10分） </li><li>蒸留水で3回洗浄</li><li> 5分間チオ硫酸ナトリウムと脅威</li><li>蒸留水で洗う</li><li> Safrinin Oとの対比</li><li>キシレンでクリア</li><li>マウント</li><li> 結果 <ol><li> Mineralised骨=黒</li><li>類骨=赤/ピンク</li></ol></li></ol></li><li>マッソンゴールドナーのトリクローム<ol class="lroman"><li>アルカリ性アルコールで洗浄する（80％エタノールおよび20分間25％アンモニアの10mlsの90mls） </li><li>水ですすいでください</li><li>蒸留水ですすぎ</li><li> 10分間ワイゲルトのヘマトキシリン​​で染色</li><li>蒸留水ですすぎ</li><li>ポンソー-フクシン最終的な解決策で5分間染色</li><li> 1％酢酸で15秒リンス</li><li>リンモリブデン酸、オレンジG液5分で染色</li><li> 1％酢酸で15秒リンス</li><li>薄緑の5分で染色</li><li> 1％酢酸で3変更をすすぐ</li><li>蒸留水ですすぎ</li><li>マウント</li><li> 結果 <ol><li> Mineralised骨=緑</li><li>類骨=赤/オレンジ</li><li>核=ブルーグレー</li><li>軟骨=紫</li></ol></li></ol></li></ol></li><li>蛍光色素の調製： </li><li>これらは、少なくとも二週間離れて、ゆっくりと静脈内注射により投与されています。 </li><li>用量<ol class="lalpha"><li>カルセイングリーン10 mg / kgのIV </li><li>オキシテトラサイクリン50 mg / kgのIV </li><li>アリザリンコンプレクソン30 mg / kgのIV </li></ol></li><li>カルセイングリーンの調製<ol class="lalpha"><li>溶けるまでカルセイングリーン1gを1 M NaOHで滴定する。 </li><li> pHは= 7.1から7.2まで、1％NaOHで調整されます。 </li><li>フィルタリングと光から遮蔽してください</li></ol></li><li>アリザリンコンプレクソンの準備<ol class="lalpha"><li>溶けるまでアリザリンコンプレクソンの3グラムを1 M NaOHで滴定する。 </li><li> pHをpHが7.1から7.2 =まで1％のHClまたはNaOHで調整されます。 </li><li>フィルタリングと光から遮蔽してください</li></ol></li><li>オキシテトラサイクリンの準備<ol class="lalpha"><li>オキシテトラサイクリンは、既製の抗菌薬として使用可能です。調製は不要です。 </li></ol></li></ol>

<ol>
	<li>Kerr, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Atlas of Functional Histology. , 164-168 (2000).</li><li>Parfitt, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bone+histomorphometry:+standardization+of+nomenclature,+symbols,+and+units.+Report+of+the+ASBMR+Histomorphometry+Nomenclature+Committee.">Bone histomorphometry: standardization of nomenclature, symbols, and units. Report of the ASBMR Histomorphometry Nomenclature Committee.</a> J Bone Miner Res. 2, 595-610 (1987).</li><li>An, Y. H., Martin, K. l. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Handbook of histology methods for bone and cartilage. , (2003).</li><li>Bancroft, J. D., Gamble, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Theory and Practice of Histological Techniques. , 352-360 (2008).</li></ol>

溶剤： <ul><li>通常の10％ホルマリン、エタノール、キシレン、ブタノール酸バッファ付き すべての組織学的グレード、硫黄フリー。 Grale科学Pty株式会社 http://www.grale.com.au </li></ul> 鋳型： <ul><li>離れて皮をむき、プラスチック金型 Graleサイエンティフィック</li></ul> スライド： <ul><li> SUPERFROST PLUS顕微鏡のスライド ボシュロム科学Pty株式会社 <a href="http://www.lomb.com.au/">http://www.lomb.com.au</a> </li></ul> 樹脂： <ul><li> GlycolMethacrylate Technovit 7100 ヘレウスKulzer <a href="http://www.kulzer-technik.de/168/Products/Histology/Technovit_7100.htm">http://www.kulzer-technik.de/168/Products/Histology/Technovit_7100.htm</a> </li><li>バッキングブロック樹脂Technovit 3040ヒストグラムブロック ヘレウスKulzer </li></ul> 汚れ （Grale科学から試薬） ゴールドナーのトリクローム染色</li> 1。ワイゲルトの鉄hemotoxylin（DeGroat） <table style="margin-left:40px;" border="0"><tr><td colspan="2" style="font-weight:bold;"> A）Hemotoxylinソリューション</td></tr><tr><td> Hemotoxylin </td><td> 1グラム</td></tr><tr><td>無水アルコール</td><td> 100mLの</td></tr><tr><td colspan="2" style="font-weight:bold;"> B）鉄溶液</td></tr><tr><td> 30％水性塩化第二鉄</td><td> 4mlの</td></tr><tr><td>塩酸（濃縮） </td><td> 1mLの</td></tr><tr><td>蒸留水</td><td> 95mL </td></tr></table>染色が使用されてすぐに前に濾過し、hemotoxylinの等量に追加。 2。ポンソー-フクシン- azophloxin株式ソリューション<table style="margin-left:40px;" border="0"><tr><td colspan="2" style="font-weight:bold;"> A）ポンソーデキシリジンソリューション</td></tr><tr><td>ポンソーデキシリジン</td><td> 0.75グラム</td></tr><tr><td>酸fuchsion </td><td> 0.25グラム</td></tr><tr><td>酢酸</td><td> 1mLの</td></tr><tr><td colspan="2">ミックス、および追加蒸留水（100ml） </td></tr><tr><td colspan="2" style="font-weight:bold;"> B）azophloxinソリューション</td></tr><tr><td> Azophloxin </td><td> 0.5グラム</td></tr><tr><td>酢酸</td><td> 0.6mL </td></tr><tr><td colspan="2">ミックス、および追加蒸留水（100ml） </td></tr><tr><td colspan="2" style="font-weight:bold;"> C）決勝は、染色液を進め</td></tr><tr><td>ポンソー-フクシン液</td><td> 5 - 10mLを</td></tr><tr><td> Azophloxin </td><td> 2mLの</td></tr><tr><td> 0.2パーセント酢酸溶液</td><td> 88mL </td></tr></table> 3。ライトグリーンソリューション<table style="margin-left:40px;" border="0"><tr><td>ライトグリーン</td><td> 1グラム</td></tr><tr><td>酢酸</td><td> 1mLの</td></tr><tr><td colspan="2">ミックス、および蒸留水を（500ミリリットル）に追加</td></tr></table> 4。 Phophomolybdic酸オレンジGソリューション<table style="margin-left:40px;" border="0"><tr><td>リンモリブデン酸</td><td> 3グラム</td></tr><tr><td>オレンジG </td><td> 2グラム</td></tr><tr><td colspan="2">蒸留水の500ミリリットルに溶解し、結晶チモールを追加。 </td></tr></table>フォンコッサ染色<table style="margin-left:40px;" border="0"><tr><td> 1％水溶液硝酸銀</td></tr><tr><td> 2.5％チオ硫酸ナトリウム</td></tr><tr><td> 1パーセントサフラニンO </td></tr></table>アルシアンブルー染色<table style="margin-left:40px;" border="0"><tr><td>アルシアンブルー8GX </td><td> 1グラム</td></tr><tr><td> 3％酢酸溶液を</td><td> 100mLの</td></tr></table> Hemotoxylinとエオシン染色<table style="margin-left:40px;" border="0"><tr><td colspan="2" style="font-weight:bold;"> a）のハリスのhemotoxylin </td></tr><tr><td> Hemotoxylin </td><td> 2.5グラム</td></tr><tr><td>絶対アルコール</td><td> 25mL </td></tr><tr><td>カリウムミョウバン</td><td> 50グラム</td></tr><tr><td>蒸留水</td><td> 500mLに</td></tr><tr><td>酸化水銀</td><td> 1.25グラムOR </td></tr><tr><td>ナトリウムヨウ素酸</td><td> 0.5グラム</td></tr><tr><td>氷酢酸</td><td> 20mlの</td></tr><tr><td colspan="2" style="font-weight:bold;"> B）エオシン液</td></tr><tr><td colspan="2">市販のエオシンを使用した。 </td></tr></table>すべての染色色素粉末は、ガー色素粉末である。 すべての試薬は分析用試薬であり、ではなくラボ試薬 蛍光色素 <table border="1" cellspacing="0" cellpadding="0"><tr><td> メーカー</td><td>蛍光色素</td><td>カタログ番号</td></tr><tr><td> Sigma - Aldrich社、シドニー</td><td>カルセイングリーン</td><td> C0875 </td></tr><tr><td> Sigma - Aldrich社、シドニー</td><td>アリザリンコンプレクソン</td><td> A3882 </td></tr><tr><td>インターオーストラリア、ビクトリア</td><td>オキシテトラサイクリン （棚抗菌薬オフ） </td><td>商品名; Engemycin 100 </td></tr></table>

undecalcified bone preparation for histology, histomorphometry and fluorochrome analysis

Undecalcified骨組織は、骨のマイクロアーキテクチャを示しています。それは、骨代謝や骨形成と骨吸収に関する重要な情報を提供する骨の鉱化と細胞成分の両方を示しています。これは、臨床と研究のさまざまなアプリケーションにおいて非常に大きな重要性を持っています。それは、美しい画像1を得られますし、そのような蛍光色素の評価や組織形態計測2のようなテクニックが可能になります。蛍光色素の分析ではカルシウムに結合する蛍光色素がその時点で鉱化量の定量化を可能にする特定の時点、で注入されている技術です。組織形態計測は、顕微鏡レベルで骨の定量化のプロセスです。 undecalcified骨の組織学を実行すると、特に大きなサイズの標本で、技術的に困難です。それは組織学を埋め込まれた標準的なパラフィンで使用されているものから技術の変化を必要とします。このビデオでは、良質のセクションを生成するプロセスを示していますし、それらを克服することで技術的な問題とメソッドを示しています。試料の準備、固定と処理が他の軟部組織と同様の方法で達成されている、しかし密度に起因すると骨の低透過性がかなり長く固定して、処理時間が必要とされる、多くの場合、数週間服用。埋め込みは、メタクリレート系樹脂などの骨に似てたり、同じ硬さと密度との支持体を使用して達成が、パラフィン浸透および埋め込みとは異なり、これは不可逆的な段階です。切片は、蛍光分析などの研究に最適な厚いセクションを、産生する研削することによって達成することができます。これは、最善のmacrotomeにダイヤモンドブレードを使用して実現されます。また、薄い切片を光学顕微鏡のために製造することができ、これは非常に鋭い刃でそりのミクロトームを使用して実現されます。そりミクロトームは、大規模な、ハードブロックに必要な追加の強度と安定性を提供します。樹脂埋込みのセクションは、実証されている汚れの様々な、で染色することができます。

Watch this Scientific Journal Video about Undecalcified Bone Preparation for Histology, Histomorphometry and Fluorochrome Analysis at JoVE.com

Undecalcified Bone Preparation for Histology, Histomorphometry and Fluorochrome Analysis

Undecalcified骨の組織学、臨床と研究のさまざまなアプリケーションのための重要な情報を提供します。それは、特に大きなサイズの標本で、技術的に困難です。このビデオでは、良質のセクションを生成するプロセスを示していますし、それらを克服することで技術的な問題とメソッドを示しています。

組織学、組織形態計測および蛍光色素分析のためのUndecalcified骨の準備

Undecalcified bone histology demonstrates the micro-architecture of bone. It shows both the mineralised and cellular components of bone, which provides ...

undecalcified-bone-preparation-for-histology-histomorphometry

Research

JoVE Journal

Biology

27.1K ビュー.  Monash University. Undecalcified骨の組織学、臨床と研究のさまざまなアプリケーションのための重要な情報を提供します。それは、特に大きなサイズの標本で、技術的に困難です。このビデオでは、良質のセクションを生成するプロセスを示していますし、それらを克服することで技術的な問題とメソッドを示しています。

ビデオ: Undecalcified Bone Preparation for Histology, Histomorphometry and Fluorochrome Analysis

The Preparation of Primary Hematopoietic Cell Cultures From Murine Bone Marrow for Electroporation

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser MXcell  electroporation system and Gene Pulser  electroporation buffer were specifically developed to transfect nucleic acids into mammalian cells and difficult-to-transfect cells, such as primary and stem cells.This video demonstrates how to establish primary hematopoietic cell cultures from murine bone marrow, and then prepare them for electroporation in the MXcell system. We begin by isolating femur and tibia. Bone marrow from both femur and tibia are then harvested and cultures are established. Cultured bone marrow cells are then transfected and analyzed.

This procedure describes how to establish primary hematopoietic cell cultures from murine bone marrow and is followed by transfection using the Gene Pulser MXCell electroporation system.

エレクトロポレーションのためのマウス骨髄からの一次造血細胞培養の準備

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser ...

生物学

A Neuronal and Astrocyte Co-Culture Assay for High Content Analysis of Neurotoxicity

High Content Analysis (HCA) assays combine cells and detection reagents with automated imaging and powerful image analysis algorithms, allowing measurement of multiple cellular phenotypes within a single assay. In this study, we utilized HCA to develop a novel assay for neurotoxicity. Neurotoxicity assessment represents an important part of drug safety evaluation, as well as being a significant focus of environmental protection efforts. Additionally, neurotoxicity is also a well-accepted in vitro marker of the development of neurodegenerative diseases such as Alzheimer's and Parkinson's diseases. Recently, the application of HCA to neuronal screening has been reported. By labeling neuronal cells with &beta;III-tubulin, HCA assays can provide high-throughput, non-subjective, quantitative measurements of parameters such as neuronal number, neurite count and neurite length, all of which can indicate neurotoxic effects. However, the role of astrocytes remains unexplored in these models. Astrocytes have an integral role in the maintenance of central nervous system (CNS) homeostasis, and are associated with both neuroprotection and neurodegradation when they are activated in response to toxic substances or disease states. GFAP is an intermediate filament protein expressed predominantly in the astrocytes of the CNS. Astrocytic activation (gliosis) leads to the upregulation of GFAP, commonly accompanied by astrocyte proliferation and hypertrophy. This process of reactive gliosis has been proposed as an early marker of damage to the nervous system. The traditional method for GFAP quantitation is by immunoassay. This approach is limited by an inability to provide information on cellular localization, morphology and cell number. We determined that HCA could be used to overcome these limitations and to simultaneously measure multiple features associated with gliosis - changes in GFAP expression, astrocyte hypertrophy, and astrocyte proliferation - within a single assay. In co-culture studies, astrocytes have been shown to protect neurons against several types of toxic insult and to critically influence neuronal survival. Recent studies have suggested that the use of astrocytes in an in vitro neurotoxicity test system may prove more relevant to human CNS structure and function than neuronal cells alone. Accordingly, we have developed an HCA assay for co-culture of neurons and astrocytes, comprised of protocols and validated, target-specific detection reagents for profiling &beta;III-tubulin and glial fibrillary acidic protein (GFAP). This assay enables simultaneous analysis of neurotoxicity, neurite outgrowth, gliosis, neuronal and astrocytic morphology and neuronal and astrocytic development in a wide variety of cellular models, representing a novel, non-subjective, high-throughput assay for neurotoxicity assessment. The assay holds great potential for enhanced detection of neurotoxicity and improved productivity in neuroscience research and drug discovery.

This article describes a novel protocol and reagent set designed for sensitive measurement of neurotoxic effects of compounds and treatments on co-cultures of neurons and astrocytes using high content analysis. Results demonstrate that high content analysis represents an exciting novel technology for neurotoxicity assessment.

神経毒性の高いコンテンツ分析のための神経とアストロサイト共培養アッセイ

High Content Analysis (HCA) assays combine cells and detection reagents with automated imaging and powerful image analysis algorithms, allowing ...

Preparation of Adult Drosophila Eyes for Thin Sectioning and Microscopic Analysis

Drosophila has long been used as model system to study development, mainly due to the ease with which it is genetically tractable. Over the years, a plethora of mutant strains and technical tricks have been developed to allow sophisticated questions to be asked and answered in a reasonable amount of time. Fundamental insight into the interplay of components of all known major signaling pathways has been obtained in forward and reverse genetic Drosophila studies. The fly eye has proven to be exceptionally well suited for mutational analysis, since, under laboratory conditions, flies can survive without functional eyes. Furthermore, the surface of the insect eye is composed of some 800 individual unit eyes (facets or ommatidia) that form a regular, smooth surface when looked at under a dissecting microscope. Thus, it is easy to see whether a mutation might affect eye development or growth by externally looking for the loss of the smooth surface ('rough eye' phenotype; Fig. 1) or overall eye size, respectively (for examples of screens based on external eye morphology see e.g.1). Subsequent detailed analyses of eye phenotypes require fixation, plastic embedding and thin-sectioning of adult eyes.
The Drosophila eye develops from the so-called eye imaginal disc, a bag of epithelial cells that proliferate and differentiate during larval and pupal stages (for review see e.g. 2). Each ommatidium consists of 20 cells, including eight photoreceptors (PR or R-cells; Fig. 2), four lens-secreting cone cells, pigment cells ('hexagon' around R-cell cluster) and a bristle. The photoreceptors of each ommatidium, most easily identified by their light sensitive organelles, the rhabdomeres, are organized in a trapezoid made up of the six "outer" (R1-6) and two "inner" photoreceptors (R7/8; R8 [Fig. 2] is underneath R7 and thus only seen in sections from deeper areas of the eye). The trapezoid of each facet is precisely aligned with those of its neighbors and the overall anteroposterior and dorsoventral axes of the eye (Fig. 3A). In particular, the ommatidia of the dorsal and ventral (black and red arrows, respectively) halves of the eye are mirror images of each other and correspond to two chiral forms established during planar cell polarity signaling (for review see e.g. 3).
The method to generate semi-thin eye sections (such as those presented in Fig. 3) described here is slightly modified from the one originally described by Tomlinson and Ready4. It allows the morphological analysis of all cells except for the transparent cone cells. In addition, the pigment of R-cells (blue arrowheads in Fig. 2 and 3) can be used as a cell-autonomous marker for the genotype of a R-cell, thus genetic requirements of genes in a subset of R-cells can readily be determined5,6.

Preparation of Adult Drosophila Eyes for Thin Sectioning and Microscopic Analysis

A standard approach to prepare adult Drosophila eyes for semi-thin sectioning and light microscopic analysis is presented here. The protocol can be used for gross morphological analysis of eye defects, or with the indicated adjustments can be used to determine genetic requirements of genes in specific cell types of the eye (e.g. clonal analysis of photoreceptors) or for electron microscopic analysis.

大人の準備ショウジョウバエ</emシンセクショニングおよび顕微鏡分析のための>アイズ

Drosophila has long been used as model system to study development, mainly due to the ease with which it is genetically tractable. Over the years, a ...

RNA In situ Hybridization in Whole Mount Embryos and Cell Histology Adapted for Marine Elasmobranchs

Marine elasmobranchs are valued animal models for biomedical and genomic studies as they are the most primitive vertebrates to have adaptive immunity and have unique mechanisms for osmoregulation 1-3. As the most primitive living jawed-vertebrates with paired appendages, elasmobranchs are an evolutionarily important model, especially for studies in evolution and development. Marine elasmobranchs have also been used to study aquatic toxicology and stress physiology in relationship to climate change 4. Thus, development and adaptation of methodologies is needed to facilitate and expand the use of these primitive vertebrates to multiple biological disciplines. Here I present the successful adaptation of RNA whole mount in situ hybridization and histological techniques to study gene expression and cell histology in elasmobranchs.
Monitoring gene expression is a hallmark tool of developmental biologists, and is widely used to investigate developmental processes 5. RNA whole mount in situ hybridization allows for the visualization and localization of specific gene transcripts in tissues of the developing embryo. The expression pattern of a gene's message can provide insight into what developmental processes and cell fate decisions a gene may control. By comparing the expression pattern of a gene at different developmental stages, insight can be gained into how the role of a gene changes during development.
While whole mount in situ's provides a means to localize gene expression to tissue, histological techniques allow for the identification of differentiated cell types and tissues. Histological stains have varied functions. General stains are used to highlight cell morphology, for example hematoxylin and eosin for general staining of nuclei and cytoplasm, respectively. Other stains can highlight specific cell types. For example, the alcian blue stain reported in this paper is a widely used cationic stain to identify mucosaccharides. Staining of the digestive tract with alcian blue can identify the distribution of goblet cells that produce mucosaccharides. Variations in mucosaccharide constituents on short peptides distinguish goblet cells by function within the digestive tract 6. By using RNA whole mount in situ's and histochemical methods concurrently, cell fate decisions can be linked to gene-specific expression.
Although RNA in situ's and histochemistry are widely used by researchers, their adaptation and use in marine elasmobranchs have met limited and varied success. Here I present protocols developed for elasmobranchs and used on a regular basis in my laboratory. Although further modification of the RNA in situ's hybridization method may be needed to adapt to different species, the protocols described here provide a strong starting point for researchers wanting to adapt the use of marine elasmobranchs to their scientific inquiries.

RNA In situ Hybridization in Whole Mount Embryos and Cell Histology Adapted for Marine Elasmobranchs

By combining methods for RNA whole mount in situ hybridization and histology, gene expression can be linked with cell fate decisions in the developing embryo. These methods have been adapted to marine elasmobranchs and facilitate the use of these animals as model organisms for biomedical, toxicology and comparative studies.

RNAを<em&gt;その場</emホールマウント胚および細胞組織学で&gt;ハイブリダイゼーションは、海洋軟骨魚類に適合した

Marine  elasmobranchs are valued animal models for biomedical and genomic studies as  they are the most primitive vertebrates to have adaptive immunity ...

Bone Conditioned Medium: Preparation and Bioassay

Autologous bone grafts are widely used in oral and maxillofacial surgery, orthopedics, and traumatology. Autologous bone grafts not only replace missing bone, they also support the complex process of bone regeneration. This favorable behavior of autografts is attributed to the three characteristics: osteoconductivity, osteogenicity, and osteoinductivity. However, there is another aspect: Bone grafts release a myriad of molecules, including growth factors, which can target mesenchymal cells involved in bone regeneration. The paracrine properties of bone grafts can be studied in vitro by the use of bone-conditioned medium (BCM). Here we present a protocol on how to prepare bone-conditioned medium from native pig cortical bone, and bone that underwent thermal processing or demineralization. Cells can be directly exposed to BCM or seeded onto biomaterials, such as collagen membranes, previously soaked with BCM. We give examples for in vitro bioassays with mesenchymal cells on the expression of TGF-&#946; regulated genes. The presented protocols should encourage to further reveal the paracrine effects of bone grafts during bone regeneration and open a path for translational research in the broad field of reconstructive surgery.

We describe here how to prepare bone-conditioned medium (BCM) and test its activity in vitro.

骨馴化培地：調製およびバイオアッセイ

Autologous bone grafts are widely used in oral and maxillofacial surgery, orthopedics, and traumatology. Autologous bone grafts not only replace missing ...

Sample Preparation for Mass Cytometry Analysis

Mass cytometry utilizes antibodies conjugated with heavy metal labels, an approach that has greatly increased the number of parameters and opportunities for deep analysis well beyond what is possible with conventional fluorescence-based flow cytometry. As with any new technology, there are critical steps that help ensure the reliable generation of high-quality data. Presented here is an optimized protocol that incorporates multiple techniques for the processing of cell samples for mass cytometry analysis. The methods described here will help the user avoid common pitfalls and achieve consistent results by minimizing variability, which can lead to inaccurate data. To inform experimental design, the rationale behind optional or alternative steps in the protocol and their efficacy in uncovering new findings in the biology of the system being investigated is covered. Lastly, representative data is presented to illustrate expected results from the techniques presented here.

This article describes the collection and processing of samples for mass cytometry analysis.

質量サイトメトリー分析のための試料調製

Mass cytometry utilizes antibodies conjugated with heavy metal labels, an approach that has greatly increased the number of parameters and opportunities ...

An Alternative Approach for Sample Preparation with Low Cell Number for TEM Analysis

Transmission electron microscopy (TEM) provides details of the cellular organization and ultrastructure. However, TEM analysis of rare cell populations, especially cells in suspension such as hematopoietic stem cells (HSCs), remains limited due to the requirement of a high cell number during sample preparation. There are a few cytospin or monolayer approaches for TEM analysis from scarce samples, but these approaches fail to get significant quantitative data from the limited number of cells. Here, an alternative and novel approach for sample preparation in TEM studies is described for rare cell populations that enables quantitative analysis.
A relatively low cell number, i.e., 10,000 HSCs, was successfully used for TEM analysis compared to the millions of cells typically used for TEM studies. In particular, Evans blue staining was performed after paraformaldehyde-glutaraldehyde (PFA-GA) fixation to visualize the tiny cell pellet, which facilitated embedding in agarose. Clusters of numerous cells were observed in ultra-thin sections. The cells had a well preserved morphology, and the ultra-structural details of the Golgi complex and several mitochondria were visible. This efficient, easy and reproducible protocol allows sample preparation from a low cell number and can be used for qualitative and quantitative TEM analysis on rare cell populations from limited biological samples.

Here, we present a protocol to prepare samples with low cell numbers for transmission electron microscopy (TEM) analysis.

TEM分析のための低細胞数とサンプル調製のための代替的アプローチ

Transmission electron microscopy (TEM) provides details of the cellular organization and ultrastructure. However, TEM analysis of rare cell populations, ...

Sample Preparation for Mass-spectrometry-based Proteomics Analysis of Ocular Microvessels

The use of isolated ocular blood vessels in vitro to decipher the pathophysiological state of the eye using advanced technological approaches has greatly expanded our understanding of certain diseases. Mass spectrometry (MS)-based proteomics has emerged as a powerful tool to unravel alterations in the molecular mechanisms and protein signaling pathways in the vascular beds in health and disease. However, sample preparation steps prior to MS analyses are crucial to obtain reproducible results and in-depth elucidation of the complex proteome. This is particularly important for preparation of ocular microvessels, where the amount of sample available for analyses is often limited and thus, poses a challenge for optimum protein extraction. This article endeavors to provide an efficient, rapid and robust protocol for sample preparation from an exemplary retrobulbar ocular vascular bed employing the porcine short posterior ciliary arteries. The present method focuses on protein extraction procedures from both the supernatant and pellet of the sample following homogenization, sample cleaning with centrifugal filter devices prior to one-dimensional gel electrophoresis and peptide purification steps for label-free quantification in a liquid chromatography-electrospray ionization-linear ion trap-Orbitrap MS system. Although this method has been developed specifically for proteomics analyses of ocular microvessels, we have also provided convincing evidence that it can also be readily employed for other tissue-based samples.

Proteome characterization of ocular microvascular beds is pivotal for in-depth understanding of many ocular pathologies in humans. This study demonstrates an effective, rapid, and robust method for protein extraction and sample preparation from small blood vessels employing the porcine short posterior ciliary arteries as model vessels for mass-spectrometry-based proteomics analyses.

眼微小循環の質量分析法を用いたプロテオミクス解析の前処理

The use of isolated ocular blood vessels in vitro to decipher the pathophysiological state of the eye using advanced technological approaches has greatly ...

A Sectioning, Coring, and Image Processing Guide for High-Throughput Cortical Bone Sample Procurement and Analysis for Synchrotron Micro-CT

Bone is a dynamic and mechanically active tissue that changes in structure over the human lifespan. The products of the bone remodeling process have been studied substantially using traditional two-dimensional techniques. Recent advancements in X-ray imaging technology via desktop micro-computed tomography (&#181;CT) and synchrotron radiation micro-computed tomography (SR&#181;CT) have allowed for the acquisition of high-resolution three-dimensional (3D) scans of a larger field of view (FOV) than other 3D imaging techniques (e.g., SEM) providing a more complete picture of microscopic structures within human cortical bone. The specimen should be accurately centered within the FOV, however, to limit the appearance of streak artifacts known to impact data analysis. Previous studies have reported procurement of irregularly shaped rectilinear bone blocks that result in imaging artifacts due to uneven edges or image truncation. We have applied a geological sampling protocol (coring) to procure consistently sized cortical bone core specimens for SR&#181;CT experiments from the anterior aspect of human femora. This coring method is efficient and minimally destructive to tissue. It creates uniform cylindrical samples that decrease imaging artifacts by nature of being isometric during rotation and providing a uniform path length for X-ray beams throughout scanning. Image processing of X-ray tomographic data of cored and irregularly shaped samples confirms the potential of the technique to improve visualization and analysis of cortical bone microarchitecture. A goal of this protocol is to deliver a reliable and repeatable method for the extraction of cortical bone cores that is adaptable for various types of high-resolution bone imaging experiments. An overarching goal of the work is to create a standardized cortical bone procurement for SR&#181;CT that is affordable, consistent, and straightforward. This procedure may further be adapted by researchers in related fields who commonly evaluate hard composite materials such as in biological anthropology, geosciences, or material sciences.

We employed a geological (coring) sampling protocol to procure cortical bone specimens of uniform size for SR&#181;CT experiments from the anterior aspect of human femora. This method is minimally destructive, efficient, results in cylindrical specimens that minimize imaging artifacts from irregular sample shapes and improves microarchitectural visualization and analysis.

高スループットコルティカル骨サンプルの採取と分析のための断面化、スコアリング、画像処理ガイド

Bone is a dynamic and mechanically active tissue that changes in structure over the human lifespan. The products of the bone remodeling process have been ...

Tissue Collection and RNA Extraction from the Human Osteoarthritic Knee Joint

Osteoarthritis (OA) is a chronic and degenerative joint disease most often affecting the knee. As there is currently no cure, total knee arthroplasty (TKA) is a common surgical intervention. Experiments using primary human OA tissues obtained from TKA provide the capability to investigate disease mechanisms ex vivo. While OA was previously thought to impact mainly the cartilage, it is now known to impact multiple tissues in the joint. This protocol describes patient selection, sample processing, tissue homogenization, RNA extraction, and quality control (based on RNA purity, integrity, and yield) from each of seven unique tissues to support disease mechanism investigation in the knee joint. With informed consent, samples were obtained from patients undergoing TKA for OA. Tissues were dissected, washed, and stored within 4 h of surgery by flash freezing for RNA or formalin fixation for histology. Collected tissues included articular cartilage, subchondral bone, meniscus, infrapatellar fat pad, anterior cruciate ligament, synovium, and vastus medialis oblique muscle. RNA extraction protocols were tested for each tissue type. The most significant modification involved the method of disintegration used for low-cell, high-matrix, hard tissues (considered as cartilage, bone, and meniscus) versus relatively high-cell, low-matrix, soft tissues (considered as fat pad, ligament, synovium, and muscle). It was found that pulverization was appropriate for hard tissues, and homogenization was appropriate for soft tissues. A proclivity for some subjects to yield higher RNA integrity number (RIN) values than other subjects consistently across multiple tissues was observed, suggesting that underlying factors such as disease severity may impact RNA quality. The ability to isolate high-quality RNA from primary human OA tissues provides a physiologically relevant model for sophisticated gene expression experiments, including sequencing, that can lead to clinical insights that are more readily translated to patients.

Primary tissues obtained from patients following total knee arthroplasty provide an experimental model for osteoarthritis research with maximal clinical translatability. This protocol describes how to identify, process, and isolate RNA from seven unique knee tissues to support mechanistic investigation in human osteoarthritis.

ヒト変形性膝関節からの組織採取とRNA抽出

Osteoarthritis (OA) is a chronic and degenerative joint disease most often affecting the knee. As there is currently no cure, total knee arthroplasty ...