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Descrizione biologica
| Specificità | α-synuclein aggregate Antibody [C4F17] rileva i livelli endogeni della proteina aggregata totale di α-sinucleina. |
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| Contesto | Gli aggregati di α-sinucleina costituiscono il segno distintivo patologico delle sinucleinopatie, tra cui la malattia di Parkinson, la demenza con corpi di Lewy e l'atrofia multisistemica, formandosi attraverso la conversione conformazionale della proteina presinaptica di 140 aminoacidi α-sinucleina, nativamente non strutturata, in fibrille amiloidi tossiche ricche di foglietti β tramite polimerizzazione nucleazione-dipendente accelerata dalla fosforilazione di Ser129 e dallo stress ossidativo. L'α-sinucleina nativa contiene ripetizioni anfifatiche KTKEGV N-terminali che consentono il legame alla membrana α-elicoide e l'associazione vescicolare, il nucleo idrofobico della regione NAC centrale che guida la conversione in filamento β amiloidogenico e un dominio C-terminale acido-prolino-ricco che inibisce l'aggregazione prematura, ma gli oligomeri prefibrillari patologici acquisiscono proprietà porogene che interrompono il traffico delle vescicole sinaptiche, compromettono l'attività del complesso mitocondriale I con produzione di specie reattive dell'ossigeno e disregolazione del calcio, compromettono i meccanismi di clearance lisosomiale/proteasomica e innescano una neuroinfiammazione microgliale cronica attraverso le vie di segnalazione TLR2/4. L'α-sinucleina monomerica chaperona l'assemblaggio del complesso SNARE ottimizzando la neurotrasmissione dopaminergica pur mantenendo i pool di vescicole sinaptiche, eppure gli intermedi oligomerici solubili, piuttosto che le fibrille mature, rappresentano le principali neurotossine che mediano la degenerazione dopaminergica nigrale selettiva tramite la propagazione del misfolding basato su stampo simile ai prioni attraverso i circuiti neurali, con triplicazioni e mutazioni del gene SNCA (A53T, A30P, E46K) che accelerano drasticamente la cinetica di aggregazione nei casi familiari, mentre le sinucleinopatie sporadiche riflettono un collasso acquisito della proteostasi. |
Informazioni sullutilizzo
| Applicazione | IHC, IF | Diluizione |
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| Reattività | Mouse, Rat, Human | ||||||
| Fonte | Rabbit Monoclonal Antibody | MW | |||||
| Tampone di conservazione | PBS, pH 7.2+50% Glycerol+0.05% BSA+0.01% NaN3 | Conservazione (Dalla data di ricevimento) |
-20°C (avoid freeze-thaw cycles), 2 years | ||||
| IHC |
Experimental Protocol：
Deparaffinization/Rehydration
1. Deparaffinize/hydrate sections:
2. Incubate sections in three washes of xylene for 5 min each.
3. Incubate sections in two washes of 100% ethanol for 10 min each.
4. Incubate sections in two washes of 95% ethanol for 10 min each.
5. Wash sections two times in dH2O for 5 min each.
6.Antigen retrieval: For Citrate: Heat slides in a microwave submersed in 1X citrate unmasking solution until boiling is initiated; continue with 10 min at a sub-boiling temperature (95°-98°C). Cool slides on bench top for 30 min.
Staining
1. Wash sections in dH2O three times for 5 min each.
2. Incubate sections in 3% hydrogen peroxide for 10 min.
3. Wash sections in dH2O two times for 5 min each.
4. Wash sections in wash buffer for 5 min.
5. Block each section with 100–400 µl of blocking solution for 1 hr at room temperature.
6. Remove blocking solution and add 100–400 µl primary antibody diluent in to each section. Incubate overnight at 4°C.
7. Remove antibody solution and wash sections with wash buffer three times for 5 min each.
8. Cover section with 1–3 drops HRPas needed. Incubate in a humidified chamber for 30 min at room temperature.
9. Wash sections three times with wash buffer for 5 min each.
10. Add DAB Chromogen Concentrate to DAB Diluent and mix well before use.
11. Apply 100–400 µl DAB to each section and monitor closely. 1–10 min generally provides an acceptable staining intensity.
12. Immerse slides in dH2O.
13. If desired, counterstain sections with hematoxylin.
14. Wash sections in dH2O two times for 5 min each.
15. Dehydrate sections: Incubate sections in 95% ethanol two times for 10 sec each; Repeat in 100% ethanol, incubating sections two times for 10 sec each; Repeat in xylene, incubating sections two times for 10 sec each.
16. Mount sections with coverslips and mounting medium.
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| IF |
Experimental Protocol:
Sample Preparation
1. Adherent Cells: Place a clean, sterile coverslip in a culture dish. Once the cells grow to near confluence as a monolayer, remove the coverslip for further use.
2. Suspension Cells: Seed the cells onto a clean, sterile slide coated with poly-L-lysine.
3. Frozen Sections: Allow the slide to thaw at room temperature. Wash it with pure water or PBS for 2 times, 3 minutes each time.
4. Paraffin Sections: Deparaffinization and rehydration. Wash the slide with pure water or PBS for 3 times, 3 minutes each time. Then perform antigen retrieval.
Fixation
1. Fix the cell coverslips/spots or tissue sections at room temperature using a fixative such as 4% paraformaldehyde (4% PFA) for 10-15 minutes.
2. Wash the sample with PBS for 3 times, 3 minutes each time.
Permeabilization
1.Add a detergent such as 0.1–0.3% Triton X-100 to the sample and incubate at room temperature for 10–20 minutes.
(Note: This step is only required for intracellular antigens. For antigens expressed on the cell membrane, this step is unnecessary.)
Wash the sample with PBS for 3 times, 3 minutes each time.
Blocking
Add blocking solution and incubate at room temperature for at least 1 hour. (Common blocking solutions include: serum from the same source as the secondary antibody, BSA, or goat serum.)
Note: Ensure the sample remains moist during and after the blocking step to prevent drying, which can lead to high background.
Immunofluorescence Staining (Day 1)
1. Remove the blocking solution and add the diluted primary antibody.
2. Incubate the sample in a humidified chamber at 4°C overnight.
Immunofluorescence Staining (Day 2)
1. Remove the primary antibody and wash with PBST for 3 times, 5 minutes each time.
2. Add the diluted fluorescent secondary antibody and incubate in the dark at 4°C for 1–2 hours.
3. Remove the secondary antibody and wash with PBST for 3 times, 5 minutes each time.
4. Add diluted DAPI and incubate at room temperature in the dark for 5–10 minutes.
5. Wash with PBST for 3 times, 5 minutes each time.
Mounting
1. Mount the sample with an anti-fade mounting medium.
2. Allow the slide to dry at room temperature overnight in the dark.
3. Store the slide in a slide storage box at 4°C, protected from light.
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Riferimenti
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