CXCL12/SDF-1 Antibody [N2D21]

카탈로그 번호 F1523

인쇄

생물학적 설명

특이성

CXCL12/SDF-1 Antibody [N2D21]는 총 CXCL12/SDF-1 단백질의 내인성 수준을 검출합니다.
배경

CXCL12는 Stromal-cell-derived factor-1 (SDF-1)으로도 알려져 있으며, 배아 발달, 장기 항상성, 백혈구 이동, 줄기세포 호밍, 혈관신생 활성 및 암 전이를 포함한 수많은 생리적 과정에 관여하는 케모카인입니다. 이는 주로 신경계에서 발현되는 SDF-1α와 SDF-1β, SDF-1γ를 비롯한 여러 스플라이스 변이체로 존재합니다. CXCL12는 단량체-이량체 평형을 형성하며, 결정화되거나 수용체인 CXCR4에 결합하면 이량체 쪽으로 이동합니다. CXCL12의 주요 수용체인 CXCR4는 태아 발달에 중요하며 T-영양성 HIV-1의 보조 수용체 역할을 하며, 여기서 CXCL12는 바이러스 진입을 억제합니다. CXCL12 발현은 허혈성 조건(예: 심장 및 골격근)에서 상향 조절되며 심근 경색 후 보호 역할을 하므로 항허혈 치료의 잠재적 표적이 됩니다. 암에서 CXCL12/CXCR4 신호는 암세포 이동 및 전이를 촉진하며, CXCR4 억제는 실험 모델에서 종양 전이를 감소시키는 것으로 나타났습니다.

사용 정보

응용 IHC, IF, FCM, CyTOF-ready 희석
IHC IF FCM
1:250 1:400 1:800
반응성 Human, Mouse
출처 Mouse Monoclonal Antibody MW 10 kDa
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.
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.

참조

  • https://pubmed.ncbi.nlm.nih.gov/22462026/
  • https://pubmed.ncbi.nlm.nih.gov/19551879/

적용 데이터

IF

Selleck 검증

  • F1523-IF
    Immunofluorescent analysis of Jurakt cells using F1523 (green, 1:400), Hoechst (blue) and tubulin (Red).

IHC

Selleck 검증

  • F1523-IHC1
    Immunohistochemical analysis of formalin fixed paraffin embedded human tonsil tissue with F1523 at 1/100 dilution.