TLR4 Antibody [J3D3]

카탈로그 번호 F2122

인쇄

생물학적 설명

특이성

TLR4 Antibody [J3D3]는 총 TLR4 단백질의 내인성 수준을 인식합니다.
배경

Toll-like 수용체 4 (TLR4)는 병원체 관련 분자 패턴 (PAMPs)을 감지하는 고도로 보존된 수용체인 패턴 인식 수용체 (PRR) 계열의 일부입니다. 이는 감염에 대한 초기 방어에 필수적입니다. TLR4는 혈액 생성 세포와 비혈액 생성 세포, 즉 내피 세포, 심근 세포, 중추신경계 (CNS) 내의 세포를 포함한 세포 표면에 존재합니다. 그람 음성 박테리아 리포폴리사카라이드 (LPS)를 인식하는 것으로 잘 알려져 있지만, 조직 손상 중에 생성되는 내인성 분자에도 결합합니다. 따라서 TLR4는 감염성 및 비감염성 자극이 수렴하여 전염증 반응을 유도하는 중요한 수용체 역할을 합니다. 외부 또는 내부 리간드에 의해 유발되는 TLR4 매개 염증은 다양한 급성 및 만성 질환에서 중요한 역할을 하며, 염증 반응의 핵심 증폭기로 작용합니다. 

사용 정보

응용 IHC, IF, FCM 희석
IHC IF FCM
1:800 1:100 1:50-1:200
반응성 Human, Mouse, Rat, Pig
출처 Mouse Monoclonal Antibody MW 120-140 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.
 
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/27293318/

적용 데이터

IF

Selleck 검증

  • F2122-IF
    Immunofluorescent analysis of Hela cells using F2122 (green, 1:200), Hoechst (blue) and tubulin (Red).

IHC

Selleck 검증

  • F2122-IHC1
    Immunohistochemical analysis of formalin fixed paraffin embedded human stomach tissue with F2122 at 1/600 dilution.