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인용 방법 1. 본문 인용 (재료 및 방법): 2. 주요 자원 표: |
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무료 전화: (877) 796-6397 -- 미국 및 캐나다 전용 -- |
팩스: +1-832-582-8590 주문: +1-832-582-8158 |
기술 지원: +1-832-582-8158 Ext:3 이메일에 주문 번호를 기재해 주십시오. 모든 이메일 문의는 영업일 기준 1일 이내에 답변해 드리기 위해 노력하고 있습니다. |
생물학적 설명
| 특이성 | α-synuclein aggregate Antibody [C4F17]는 총 α-시누클레인 응집 단백질의 내인성 수준을 검출합니다. |
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| 배경 | α-시누클레인 응집체는 파킨슨병, 레비 소체 치매 및 다계통 위축을 포함한 시누클레인병증의 특징적인 병리학적 특징을 구성하며, 세린129 인산화 및 산화 스트레스에 의해 가속화되는 핵 형성 의존적 중합을 통해 원래 구조화되지 않은 140-아미노산 시냅스 전 단백질 α-시누클레인의 독성 β-시트 풍부 아밀로이드 섬유로의 형태 변화를 통해 형성됩니다. 천연 α-시누클레인은 α-나선형 막 결합 및 소포 연합을 가능하게 하는 N-말단 양친매성 KTKEGV 반복 서열, 아밀로이드 형성 β-가닥 전환을 유도하는 중앙 NAC 영역의 소수성 코어, 그리고 조기 응집을 억제하는 C-말단 산성-프롤린 풍부 도메인을 포함하지만, 병리학적 전섬유성 올리고머는 시냅스 소포 수송을 방해하고, 활성 산소 종 생성 및 칼슘 조절 이상을 동반한 미토콘드리아 복합체 I 활성을 손상시키며, 리소좀/프로테아좀 제거 메커니즘을 손상시키고, TLR2/4 신호 경로를 통해 만성 미세아교세포 신경염증을 유발하는 공극 형성 특성을 획득합니다. 단량체 α-시누클레인은 SNARE 복합체 조립을 촉매하여 시냅스 소포 풀을 유지하면서 도파민 신경전달을 최적화하지만, 성숙한 섬유보다는 가용성 올리고머 중간체가 프리온 유사 템플릿화된 오접힘 전파를 통해 신경 회로 전반에 걸쳐 선택적 흑질 도파민성 퇴행을 매개하는 주요 신경독으로 작용하며, SNCA 유전자 삼중복 및 돌연변이(A53T, A30P, E46K)는 가족성 사례에서 응집 동역학을 극적으로 가속화하는 반면, 산발성 시누클레인병증은 후천적 단백질 항상성 붕괴를 반영합니다. |
사용 정보
| 응용 | IHC, IF | 희석 |
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| 반응성 | Mouse, Rat, Human | ||||||
| 출처 | Rabbit Monoclonal Antibody | MW | |||||
| 보관 완충액 | PBS, pH 7.2+50% Glycerol+0.05% BSA+0.01% NaN3 | 보관 (수령일로부터) |
-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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참조
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