複雑な脳の地形を明らかにするためWholemount免疫組織化学
Summary
神経回路は、地形的に特定の分子プロファイルを持つ機能的なコンパートメントに編成されています。ここでは、多彩なwholemount免疫組織化学的染色のアプローチを使用してグローバル脳の地形を明らかにするための実用的かつ技術的な手順を提供しています。我々はよく理解され細胞構築、小脳の回路を用いる方法の有用性を示しています。
Abstract
小脳の繰り返し、十分に理解細胞のアーキテクチャでは、脳の地形を探索するのに理想的なモデルシステムを確認します。比較的均一な細胞構築の基礎となると、遺伝子およびタンパク質発現の矢ドメインの複雑な配列です。小脳の分子区画は、求心性線維の解剖学的および機能的な組織によってミラーリングされています。完全に小脳組織の複雑さを理解するために我々は、以前はマウス小脳におけるパターン欠陥のハイスループット分析のためのwholemount染色アプローチを洗練された。このプロトコルは、詳細で正常にwholemount免疫染色を用いて成体マウス小脳におけるタンパク質の発現パターンを明らかにするために有用である試薬、ツール、および実用的な手順について説明します。手順はここで脳の微細な地形は、そのに明らかにすることができる方法の例としてzebrinII / aldolaseCの式を使用してこの方法の有用性を示す強調表示されたネイティブの三次元立体配座。についても説明し、分子の地形の比較研究のための求心性の予測および大型小脳におけるタンパク質発現の可視化を可能にするプロトコルへの適応があります。これらのアプリケーションを説明するために、ラット小脳の求心性染色からのデータが含まれています。
Protocol
1。動物の灌流および小脳の解剖蛋白質に応じて、灌流が成功した染色1,2のために不可欠である可能性があります。経心臓的に灌流は麻酔薬の適切な使用を必要とする侵襲的、非生存の手順です。正しいトレーニング、制度の承認、およびIACUC承認はすべての手順を実行する前に必要です。それは、常に実験的な要件を識別し、適切なトレーニングを取得するにヘルプを表示す…
Discussion
我々の開発および成人の脳内タンパク質の発現を明らかにするための多彩な免疫組織化学的アプローチを用いて成功したwholemount染色するために必要な技術的な詳細を説明してきました。このアプローチを使用することにより、複雑な分子の発現パターンを解析することができ、脳の地形は、手間と時間がかかり、組織切片の手順を必要とせずに感謝しています。
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Disclosures
The authors have nothing to disclose.
Acknowledgements
RVSはイェシーバー大学のアルバート·アインシュタイン医科大学からのスタートアップ資金を新たな捜査官でサポートされています。
Materials
| Materials | Function in protocol |
| Perfusion pump (Fisher Scientific/13-876-2) | Allows for consistent and slow perfusion. |
| Sharp-tip Scissors (FST/14081-08) | General use in perfusion and dissection. |
| Blunt-tip Forceps (FST/91100-12) | To stabilize the heart for insertion of the perfusion needle. |
| Forceps (FST by Dumont AA/11210-10) | For use during dissection of the brain from the skull and to separate the cerebellum from the rest of the brain. These are essential because they have a slightly rounded tip that helps minimize damage to the cerebellum during dissection. |
| Nutator (Fisher Scientific) | Used to keep tissue in motion during incubation periods. |
| 1.5 mL tube (Sarstedt/Screw Cap Micro Tube) | All steps of the histochemistry protocol take place in these microtubes. The rounded bottom ensures that the cerebellum stays in motion. |
| Perforated spoon (FST/10370-17) | Used to keep wholemounts in the microtubes while gently decanting out the spent solution. |
| Leica MZ16 FA microscope | Used to examine wholemount staining. |
| Leica DFC3000 FX camera | Used to capture wholemount images. |
Table 1.
| Example calendar for a typical wholemount experiment | ||||||||
| Day 1 | Dent’s fix, room temperature, 8 hrs | Dent’s bleach, 4°C, overnight | ||||||
| Day 2 | 100% MeOH, room temperature, 2x, 30 min each | 100% MeOH, Freeze/thaw, 4x, 30 min/15 min |
100% MeOH, -80°C, overnight | |||||
| Day 3 | 50% MeOH/50% PBS, room temperature, 60-90 min | 15% MeOH/ 85% PBS, room temperature, 60-90 min | 100% PBS, room temperature, 60-90 min | 10μg/mL Proteinase K in PBS, room temperature, 2-3 min | 100% PBS, room temperature, 3x, 10 min each | PMT, 4°C, overnight | ||
| Day 4-5 | PMT + 1° antibody + 5% DMSO, 4°C, 48 hrs | |||||||
| Day 6 | PMT, 4°C, 2-3x, 2-3 hrs each | PMT + 2° antibody + 5% DMSO, 4°C, 24 hours (Or begin amplification steps with ABC complex) | ||||||
| Day 7 | PMT, 4°C, 2-3x, 2-3 hrs each | PBT, room temperature, 2 hrs | Incubate in fresh DAB in PBS until optimal staining is visualized | |||||
Table 2.
| Recipes (*=prepare fresh every time) | |
| PBS (phosphate buffered saline) | 0.1M phosphate buffered saline in deionized water. pH 7.2 (Sigma tablets; P4417) |
| PFA (Paraformaldehyde) | Made and stored frozen as a 20% solution and then diluted to 4% in PBS for the working solution (Fisher Scientific; T353) |
| Dent’s Fixative3* | 4 parts methanol 1 part dimethylsulfoxide (DMSO; Fisher Scientific; D159-4) |
| Dent’s Bleach3* | 4 parts methanol 1 part dimethylsulfoxide (DMSO; Fisher Scientific; D159-4) 1 part 30% hydrogen peroxide |
| Enzymatic Digestion | 10 μg/ml of Proteinase K (Roche Diagnostics; 03115828001) in PBS. |
| PBST | PBS containing: 0.1% Tween-20 (Fisher Scientific, BP337; Triton can also be used in place of Tween-20 in all instances.) |
| PMT25* | PBS containing: 2% nonfat skim milk powder (Carnation preferred) 0.1% Tween-20 (Fisher Scientific; BP337) |
| PBT25* | PBS containing: 0.2% bovine serum albumin (Sigma; B9001S) 0.1% Tween-20 (Fisher Scientific; BP337) |
| DAB* | Dissolve one 10-mg tablet of 3,3-diaminobenzidine (Sigma-Aldrich; D5905) in 40 ml of PBS. Add 10 μl of 30% hydrogen peroxide to initiate reaction). |
| ABC Complex Solution | Vectastain kit (Vector laboratories, Inc; PK-4000) |
Table 3.
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Tags
Wholemount ImmunohistochemistryBrain TopographyCerebellumCellular ArchitectureGene Expression DomainsProtein Expression DomainsMolecular CompartmentalizationAfferent FibersPatterning DefectsMouse CerebellumWholemount Staining ApproachProtein Expression PatternsZebrinII/aldolaseC ExpressionThree-dimensional ConformationAfferent ProjectionsComparative StudiesMolecular TopographyRat Cerebellum
Cite This Article
White, J. J., Reeber, S. L., Hawkes, R., Sillitoe, R. V. Wholemount Immunohistochemistry for Revealing Complex Brain Topography. J. Vis. Exp. (62), e4042, doi:10.3791/4042 (2012).