Wholemount Immunhistokemi for at afsløre Complex Brain Topografi
Summary
Neurale kredsløb topografisk organiseret i funktionelle rum med specifikke molekylære profiler. Her giver vi de praktiske og tekniske skridt til at afsløre den globale hjerne topografi med et alsidigt wholemount immunhistokemisk farvning tilgang. Vi demonstrerer anvendeligheden af metoden under anvendelse af velforståede cytoarchitecture og kredsløb cerebellum.
Abstract
Den gentagne og godt forstået cellulære arkitektur af lillehjernen gør det til en ideel model for at udforske hjernen topografi. Underliggende dets relativt ensartet cytoarchitecture er en kompleks række af parasagittal domæner genet og proteinekspression. Den molekylære opdeling af lillehjernen er afspejlet i den anatomiske og funktionelle organisering af afferente fibre. For fuldt ud at forstå kompleksiteten af cerebellare organisation, vi tidligere har forfinet en wholemount farvning tilgang til high throughput analyse af mønstergivende fejl i musen lillehjernen. Denne protokol beskriver i detaljer de reagenser, værktøjer og praktiske skridt, som er nyttige at kunne afsløre protein ekspressionsmønstre i den voksne mus lillehjernen ved hjælp wholemount immunfarvning. Trinene fremhævet her demonstrere anvendeligheden af denne metode at bruge udtryk zebrinII / aldolaseC som et eksempel på, hvordan den fine topografi i hjernen, kan blive afsløret i sinnative tredimensionelle konformation. Også beskrevet er tilpasninger til den protokol, der giver mulighed for visualisering af protein udtryk i afferente fremskrivninger og store cerebella for sammenlignende studier af molekylære topografi. For at illustrere disse ansøgninger, er data fra afferent farvning af rottecerebellum inkluderet.
Protocol
Discussion
Vi har beskrevet de tekniske detaljer, der er nødvendige for en vellykket wholemount farvning ved hjælp af en alsidig immunhistokemisk tilgang til afslørende protein-ekspression i udviklende og voksne hjerne. Ved hjælp af denne fremgangsmåde, kan komplekse molekylære ekspressionsmønstre skal analyseres, og hjernen topografi værdsat uden behov for besværlige og tidskrævende væv koblinger procedurer.
Denne protokol er blevet anvendt til at afsløre den mønstrede ekspressionen af ad…
Disclosures
The authors have nothing to disclose.
Acknowledgements
RVS understøttes af nye investigator startfonde fra Albert Einstein College of Medicine i Yeshiva University.
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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