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PowerPoint Template. Table of Contents. 1. Introduction. 2. Objective. Table Of Contents. 3. Procedure. 4. Results. 5. Conclusion. 1. Introduction. H&E staining Immunostaining PCNA FITC TRITC. 1. Introduction. P C N A. Proliferating Cell Nuclear Antigen.
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Table of Contents 1. Introduction 2. Objective Table Of Contents 3. Procedure 4. Results 5. Conclusion
1. Introduction • H&E staining • Immunostaining • PCNA • FITC • TRITC
1. Introduction P C N A Proliferating Cell Nuclear Antigen co-factor for DNA polymerase delta in S-phase, DNA synthesis : temporal specificity of PCNA expression eukaryotic chromosomal DNA replication, DNA repair 세포분화 확인의 좋은 수단: G1 → S PC-10과 결합: antibody
1. Introduction F I T C Fluoresceine, Fluorescein isothiocyanate small organic molecule, typically conjugated to proteins via primary amines excited by the 488 nm line of an argon laser, emission is collected at 530 nm.
1. Introduction T R I T C tetramethylrhodamine isothiocyanate 단백질 결합 형광 염색, 여러 isomer형태로 활용
2. Objective 담당 조직의 다양한 세포 관찰 면역염색법으로 특정 단백질의 발현을 확인 실험목적 동물조직 샘플 준비방법 습득
2. Objective 실험목적 신경발생 과정에서 분열하는 신경세포조직을 mouse fetus의 neural tube에서 살펴본다 fixation, processing, section cutting, staining등 histological technique를 배운다 Immunostaining을 이용하여 원하는 단백질이 어느 조직에서 발현되는가를 식별하여 기능/패턴 유추
3. Procedure 1 FIXATION 2 H&E STAINING 3 DAB STAINING 4 FLUORESCENT STAINING PROTOCOL
3. Procedure • Slide 제작 및 현미경 관찰 • Mouse fetus의 neural tube를 확인 • hematoxylin 염색 • eosin 염색 Staining • 준비된 paraffin block5㎛ section • ethanol ▶ floating bath ▶ 저장 Section cutting • sample을 파라핀 mold에 고정 • mold를 식힌 후 4℃ 저장 Processing • 가능한 한 살아있는 형태로 조직 보존 • Day 10 fetus 껍질 제거 • 탈수▶ETOH▶xylene▶parrafin Fixation
3. Procedure Immunostaining 형광염색 파라핀제거 • Incubate FITC or TRITC-conjugated Secondary Ab diluted in Blocking sol. • Incubate slides with DAPI • Wash with PBST 1,2차 항체 • Xylene 처리 • EtOH 처리 • DW 처리 • incubate with blocking solution • incubate with primary antiserum. • wash with PBST • incubate withsecondary Ab in blocking solution • incubate with DAPI • wash with PBST 세포막처리 PBST 처리 Antibody의 침입을 용이하게
3. Procedure 관찰 및 발표 진 행 7일차 FLUORESCENT STAINING 6일차 DAB STAINING 5일차 H&E STAINING 3,4일차 MOLDING SECTIONING 1,2일차 FIXATION PROCESSING
4. Results Mouse fetus Slide # Day 10
4. Results Mouse fetus Slide # Day 10
4. Results Mouse fetus Slide # Day 10 Immunostaining DAPI FITC TRITC
4. Results Day 10 Immunostaining DAPI
4. Results Day 10 Immunostaining FITC
4. Results Day 10 Immunostaining TRITC
4. Results Day 15 H&E
4. Results Day 15 DAPI
4. Results Mouse fetus Slide # Day 15 Immunostaining DAPI FITC
4. Results Day 15 Immunostaining DAPI
4. Results Day 15 Immunostaining FITC
5. Conclusion Immunostaining `
5. Conclusion NEURAL TUBE
5. Conclusion IMMUNOSTAINING Day 10 : no signal Day 15 : no signal FAILURE
5. Conclusion 1 2 3 TARGET 조직의 분별 IMMUNOSTAINING 과정 기타 실험 과정 실패요인
5. Conclusion 1 TARGET 조직의 분별 실패요인 Mouse fetus의 neural tube 식별 실패 Day 15 sample의 경우는식별 가능 Day 15 sample도 signal 부재 조직 식별 실패 외의 요인 추정
5. Conclusion 2 IMMUNOSTAINING 과정 실패요인 지나친 blocking Antibody의 결합 실패 지나친 washing 빛에 노출
5. Conclusion 3 기타 실험 과정 실패요인 세포 고정 시간의 문제 : fixation duration 세포분열 시기의 문제 : PCNA 존재여부 실험재료의 문제 기타 실험과정 중 실수
5. Conclusion References Histological and histochemical methods : theory and practice, Kiernan, J. A. (John Alan) , 2nd ed. Oxford, England ; New York : Pergamon Press, 1990. N-CAM alterations in splotch neural tube defect mouse embryos, Connie E. Moase & Daphne G. Trasler, Development 113, 1049-1058 (1991) http://www.drmr.com/abcon/Ffspec.html http://www.chemicon.com/ http://dev.biologists.org/ http://www.ihcworld.com/_protocols/antibody_protocols/pcna_santa_cruz.htm
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