Technology and methodology of pathology research Department of Pathology Peking Union Medical College Liang Zhi-yong West China School of Medicine Sichuan University Liu Wei-ping

Technology and methodology of pathology research animal experiment animal experiment tissue and cell culture tissue and cell culture electron microscopy electron microscopy histochemistry and immunohistochemistry histochemistry and immunohistochemistry in situ hybridization ， ISH in situ hybridization ， ISH polymerase chain reaction ， PCR polymerase chain reaction ， PCR microdissection microdissection

Technology and methodology of pathology research Laser scanning confocal microscopy ， LSCM Laser scanning confocal microscopy ， LSCM Flow cytometry ， FCM Flow cytometry ， FCM Comparative genomic hybrzation ， CGH Comparative genomic hybrzation ， CGH Biochip technique Biochip technique Molecular imaging Molecular imaging Image analysis Image analysis

Electron Microscopy Transmission electron microscope ， TEM Transmission electron microscope ， TEM Scanning electron microscopy, SEM Scanning electron microscopy, SEM Ultrastructural pathology Ultrastructural pathology Freeze-etching technology Freeze-etching technology

1 ． Preparation of specimens for electron microscopy 1 ． Preparation of specimens for electron microscopy ① The tissues are fresh ， and specimens dissection are correct ② Double tissue fixation ③ Tissue embedding uses epoxy resin ④ Tissue orientation ⑤ Ultrathin section

2 ． Electron microscopy application The research and observation in embryology and histology The research and observation in embryology and histology The observation and diagnosis for subcellular structures of many diseases The observation and diagnosis for subcellular structures of many diseases Identification for the sources and features of some difficult tumors Identification for the sources and features of some difficult tumors Limitation: small samples drawn, preparation of more complex, special limited observation range, and so on. Limitation: small samples drawn, preparation of more complex, special limited observation range, and so on.

Histochemistry and Immunohistochemistry Histochemistry ： study of the chemical composition of tissues by means of specific staining reactions Histochemistry ： study of the chemical composition of tissues by means of specific staining reactions Immunohistochemistry (IHC): use of antibody- antigen stains to create reactions to locate markers specific to certain tissues or cells. Immunohistochemistry (IHC): use of antibody- antigen stains to create reactions to locate markers specific to certain tissues or cells. monoclonal antibody monoclonal antibody polyclonal antibody polyclonal antibody

1. IHC Staining laheled dextran polymer ， LDP laheled dextran polymer ， LDP labeled streptoavidin-biotin ， LSAB labeled streptoavidin-biotin ， LSAB Two-step LDP: Envision staining: Two-step LDP: Envision staining: Advantage — saving, simple, and Less interference factors Advantage — saving, simple, and Less interference factors Disadvantage — cost is higher than LSAB Disadvantage — cost is higher than LSAB

1 2 3 antigen Primary antibody Biotin second antibody Streptavidin - enzyme complex 1.add primary antibody ； 2.add biotin second antibody ； 3. add Streptavidin - enzyme complex

2. IHC Staining Technique Application The detection for expression of various protein or peptide The detection for expression of various protein or peptide Determination for cell origin and differentiation Determination for cell origin and differentiation The analysis for lymphocytes The analysis for lymphocytes immunophenotype immunophenotype The research for cell proliferation, cell cycle and signal transduction The research for cell proliferation, cell cycle and signal transduction Prognosis and targeted therapy of Cancer Prognosis and targeted therapy of Cancer

In situ hybridization ， ISH Fluorescence in situ hybridization （ FISH ） —— a process which paints chromosomes or portions of chromosomes with fluorescent molecules Fluorescence in situ hybridization （ FISH ） —— a process which paints chromosomes or portions of chromosomes with fluorescent molecules ★ Direct method ： DNA for probe use is labeled with fluorescent ★ Indirect method ： non fluorescent molecules which are then detected by fluorescent antibodies

ISH Applications mRNA transcriptional orientation 1. Cell specific mRNA transcriptional orientation 2. Virus detection (DNA or RNA) 3. Gene amplifications and deletions 4. Identifying morphology or relationships among types of organisms 5. Numerical chromosomal alterations 6. Interphase/metaphase cytogenetics

Polymerase chain reaction ， PCR In situ PCR: a technique in molecular biology to amplify a single or few copies of a piece of DNA across several orders of magnitude, generating thousands to millions of copies of a particular DNA sequence. In situ PCR: a technique in molecular biology to amplify a single or few copies of a piece of DNA across several orders of magnitude, generating thousands to millions of copies of a particular DNA sequence. in situ reverse transcription-PCR ， RT-PCR in situ reverse transcription-PCR ， RT-PCR Self sustained sequence replication reaction ， 3SR Self sustained sequence replication reaction ， 3SR

PCR technology Applications The detection and orientation for low copy endogenous gene The detection and orientation for low copy endogenous gene The detection for single copy DNA sequence The detection for single copy DNA sequence The research for gene mutation, gene rearrangement and chromosomal translocation The research for gene mutation, gene rearrangement and chromosomal translocation The detection and orientation for exogenous genes The detection and orientation for exogenous genes

Microdissection Microdissection: the extraction of selected cells from a microscopically heterogeneous sample of tissue. Microdissection: the extraction of selected cells from a microscopically heterogeneous sample of tissue. Laser capture microdissection ， LCM: use of a laser through a microscope to cause selected cells to adhere to a film. Laser capture microdissection ， LCM: use of a laser through a microscope to cause selected cells to adhere to a film.

② Microdissection pick up target cells (e.g.tumor cells) from HE slides for DNA extraction By Manual By laser

Laser scanning confocal microscopy （ LSCM ） A technique for obtaining high-resolution optical images with depth selectivity A technique for obtaining high-resolution optical images with depth selectivity Main function: widely-used in numerous biological science disciplines, from cell biology and genetics to microbiology and developmental biology Main function: widely-used in numerous biological science disciplines, from cell biology and genetics to microbiology and developmental biology

Flow cytometry ， FCM A technique for counting and examining microscopic particles. A technique for counting and examining microscopic particles. Such as cells and chromosomes, by suspending them in a stream of fluid and passing them by an electronic detection apparatus. Such as cells and chromosomes, by suspending them in a stream of fluid and passing them by an electronic detection apparatus.

1. FCM basic structure and working principle Consists of three parts ： Consists of three parts ： ① a sensing System ① a sensing System ② a computer system ② a computer system ③ Circuits, optical path and water systems ③ Circuits, optical path and water systems

Working Principle of FCM A number of detectors are aimed at the point where the stream passes through the light beam: one in line with the light beam and several perpendicular to it. A number of detectors are aimed at the point where the stream passes through the light beam: one in line with the light beam and several perpendicular to it. Fluorescent chemicals found in the particle or attached to the particle may be excited into emitting light at a longer wavelength than the light source. Fluorescent chemicals found in the particle or attached to the particle may be excited into emitting light at a longer wavelength than the light source. This combination of scattered and fluorescent light is picked up by the detectors, and, by analysing fluctuations in brightness at each detector. This combination of scattered and fluorescent light is picked up by the detectors, and, by analysing fluctuations in brightness at each detector.

2. FCM application  Analysis of cell cycle and cell proliferation kinetics  Analysis of cell proliferation and apoptosis  Analysis of cell differentiation, supporting identification of benign and malignant tumor  Sorting cells or particles  Detection of drugs in the cell content, distribution and mechanisms distribution and mechanisms

Comparative genomic hybrzation ， （ CGH ） A molecular-cytogenetic method for the analysis of copy number changes (gains/losses) in the DNA content of a given subject's DNA and often in tumor cells. A molecular-cytogenetic method for the analysis of copy number changes (gains/losses) in the DNA content of a given subject's DNA and often in tumor cells.

Biochip technique Gene chip: consists of an arrayed series of thousands of microscopic spots of DNA oligonucleotides, called features, each containing picomoles (10−12 moles) of a specific DNA sequence, known as probes. Gene chip: consists of an arrayed series of thousands of microscopic spots of DNA oligonucleotides, called features, each containing picomoles (10−12 moles) of a specific DNA sequence, known as probes. Protein chip: provides a multiplex approach to identify protein–protein interactions, to identify the substrates of protein kinases, to identify transcription factor protein-activation. Protein chip: provides a multiplex approach to identify protein–protein interactions, to identify the substrates of protein kinases, to identify transcription factor protein-activation. Tissue array: consist of paraffin blocks, separate tissue cores are assembled in array fashion to allow multiplex histological analysis. Tissue array: consist of paraffin blocks, separate tissue cores are assembled in array fashion to allow multiplex histological analysis.

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Molecular imaging Application of imaging methods, on the living condition of the biological process of cellular and molecular level of qualitative and quantitative research. Application of imaging methods, on the living condition of the biological process of cellular and molecular level of qualitative and quantitative research. This image from the non-specific changes to specific imaging for disease biology, disease, early detection, characterization, assessment and treatment have brought a major impact. This image from the non-specific changes to specific imaging for disease biology, disease, early detection, characterization, assessment and treatment have brought a major impact.

Molecular imaging application Optical in vivo imaging Optical in vivo imaging Radionuclide imaging Radionuclide imaging

Image analysis The extraction of meaningful information from images; mainly from digital images by means of digital image processing techniques. The extraction of meaningful information from images; mainly from digital images by means of digital image processing techniques. Image analysis tasks can be as simple as reading bar coded tags or as sophisticated as identifying a person from their face. Image analysis tasks can be as simple as reading bar coded tags or as sophisticated as identifying a person from their face.

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