La inmunohistoquímica wholemount de Topografía Revelando Complex Brain
Summary
Circuitos neuronales están topográficamente organizadas en compartimentos funcionales con determinados perfiles moleculares. Aquí, ofrecemos los pasos prácticos y técnicos para revelar la topografía global del cerebro mediante un método versátil wholemount tinción inmunohistoquímica. Se demuestra la utilidad del método que utiliza la citoarquitectura bien entendida y la circuitería de cerebelo.
Abstract
La arquitectura repetida y bien entendido-celular del cerebelo lo convierten en un sistema modelo ideal para explorar la topografía cerebral. Detrás de su citoarquitectura relativamente uniforme es un complejo conjunto de dominios parasagitales de expresión génica y proteica. La compartimentación molecular del cerebelo se refleja en la organización anatómica y funcional de las fibras aferentes. Para apreciar plenamente la complejidad de la organización del cerebelo que previamente refinado un enfoque tinción wholemount de análisis de alto rendimiento de los defectos de modelado en el cerebelo de ratón. Este protocolo se describe en detalle los reactivos, instrumentos y medidas prácticas que son útiles para revelar los patrones de éxito de expresión de proteínas en el cerebelo de ratones adultos mediante el uso de inmunotinción wholemount. Los pasos resaltada aquí demuestran la utilidad de este método que utiliza la expresión de zebrinII / aldolaseC como un ejemplo de cómo la topografía fino del cerebro puede ser revelada en sunativo conformación tridimensional. También se describen las adaptaciones del protocolo que permite la visualización de expresión de la proteína en las proyecciones aferentes y cerebelo, sin limitación para los estudios comparativos de la topografía molecular. Para ilustrar estas aplicaciones, los datos de tinción aferente del cerebelo de rata están incluidos.
Protocol
Discussion
Hemos descrito los detalles técnicos necesarios para la tinción wholemount éxito utilizando un enfoque de inmunohistoquímica versátil para la expresión de la proteína revelador en el desarrollo del cerebro y de adultos. Mediante el uso de este enfoque, los patrones de expresión complejos moleculares pueden ser analizados y la topografía del cerebro apreciado sin la necesidad de laborioso y procedimientos que consumen tejidos de seccionamiento.
Este protocolo se ha utilizado para rev…
Disclosures
The authors have nothing to disclose.
Acknowledgements
RVS con el apoyo de nuevo investigador de puesta en marcha de fondos de Albert Einstein College of Medicine de la Universidad Yeshiva.
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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