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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 | CD11b + CD11c Antibody [C8C21]는 총 CD11b 및 CD11c 단백질의 내인성 수준을 검출합니다. |
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| 배경 | CD11b(αM, ITGAM)와 CD11c(αX, ITGAX)는 β2-인테그린 계열(CD11/CD18)의 인테그린 α-서브유닛으로, 각각 CD11b/CD18(Mac-1, CR3) 및 CD11c/CD18(p150,95, CR4) 이종이량체를 형성하며, 주로 단핵구, 대식세포, 호중구 및 수지상 세포를 포함한 골수성 백혈구에 발현됩니다. 둘 다 7개의 날개를 가진 β-프로펠러 도메인, 허벅지 도메인, 송아지-1/송아지-2 도메인, 그리고 Asp, Ser, Glu 잔기에 의한 Mg²⁺ 배위를 포함하는 금속 이온 의존성 접착 부위(MIDAS)를 포함하는 주요 리간드 결합 부위로서 삽입된 I-도메인(폰 빌레브란트 인자 A-유사)을 가진 큰 세포외 영역을 특징으로 합니다. CD11c/CD18은 β2-꼬리에 탈린 결합에 의해 조절되는 구부러진(낮은 친화도), 확장-닫힌, 확장-열린(높은 친화도) 형태를 취하며, α-사슬 Ser1158에서의 CD11c 인산화는 활성화를 강화합니다. CD11b/CD18은 iC3b, ICAM-1, 피브리노겐에 결합하여 PI3K/Akt 신호 전달을 통해 백혈구 접착, 이주, 옵소닌화된 입자의 식세포 작용 및 호흡 폭발을 매개하며, CD11c/CD18은 유사하게 iC3b/보체 매개 식세포 작용, DC 항원 포획(예: CD47 결핍 세포) 및 내피/피브로넥틴에 대한 접착을 촉진하여 세포골격 재구성, NF-κB/MAPK 활성화 및 선천/적응 면역을 위한 사이토카인 생산을 유발합니다. 기능 이상은 만성 염증(예: 류마티스 관절염), 대식세포 침윤을 통한 고혈압 및 면역 감시 기능 저하를 통한 암 진행에 기여합니다. |
사용 정보
| 응용 | IHC, IF | 희석 |
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| 반응성 | Rat | ||||||
| 출처 | Mouse 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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