Histology Protocols

Creative Bioarray provides an easily accessible reference for histological techniques. Here, we detail the techniques for investigating the expression of many different types of targets in a variety of tissue and samples from sections and cells, covering a wide range of techniques from biospecimen treatment to tissue sections, tissue staining, and various types of immunostaining. We provide step-by-step guide to techniques including guidelines, methods and notes.

Biospecimen Collection Protocol

Classification: Biospecimen Treatment.

Guidelines: A wide variety of specimen types may be collected for storage, depending on availability and study goals, such as tissues that from autopsy and transplant, cell lines, and so on.

Biospecimen Processing Protocol

Classification: Biospecimen Treatment.

Guidelines: Specimens are processed according to the study design and the methods most appropriate for preserving the analytes of interest. For a particular specimen type and analysis, several processing methods may be appropriate.

Biospecimen Storage Protocol

Classification: Biospecimen Treatment.

Guidelines: Depending on the intended laboratory analyses, and other considerations, specimens and their aliquots may be stored under different suitable conditions.

Biospecimen Information Management Protocol

Classification: Biospecimen Treatment.

Guidelines: Driven by advances in molecular technologies, information management is critical to the molecular epidemiology research enterprise.

Fixation Protocol

Classification: Biospecimen Treatment.

Guidelines: Freshly harvested tissue of interest should be immediately fixed to avoid degradation.

Decalcification Protocol

Classification: Biospecimen Treatment.

Guidelines: Decalcification is required for processing bone tissue in routine diagnostic practice. Control of this step is crucial because it may have important consequences for establishing the diagnosis.

Decolorization Protocol

Classification: Biospecimen Treatment.

Guidelines: Decolorization refers to the process of removing brightly colored organic impurities from the sample mixture. The procedure is usually carried out in the solution phase after the solid product and impurities are dissolved in a suitable solvent.

Dehydration Protocol

Classification: Biospecimen Treatment.

Guidelines: The process of replacing water contained in tissues or cells with a dehydrating agent is called tissue dehydration.

Wax Infiltration Protocol

Classification: Biospecimen Treatment.

Guidelines: After the tissue is completely transparent with the transparent agent, it is placed in the paraffin wax which is melted at about 65°C. The process of impregnation in the electric thermostat at 65°C is called wax immersion.

Embedding & Blocking Out Protocol

Classification: Biospecimen Treatment.

Guidelines: Embedding is to remove the fixed, dehydrated, transparent and wax-soaked tissue blocks from the final wax bath and place them into the embedding frame filled with molten paraffin wax. Embedding into blocks enables the tissue and embedding agent to be fused together and cooled rapidly. This procedure is called embedding.

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Cellular Protocols

The cell is the basic structural and functional unit of life forms. Cells share many common features, yet they can look wildly different. In fact, cells have adapted over billions of years to a wide array of environments and functional roles. Creative Bioarray provides an easily accessible reference for cellular techniques from cell isolation to cell culture. Here, we provide step-by-step guide to techniques including guidelines, methods and notes.

Natural Killer Cell Culture Protocol

Classification: Cell Culture.

Guidelines: Natural killer cells are an interleukin-2 (IL-2)-dependent natural killer (NK) cell line derived from peripheral blood mononuclear cells of a 50-year-old white male with acute non-Hodgkin lymphoma. The cells are rounded or elliptic, transparent and grow in clumps.

Construction and Culture Protocol of Neural Stem Cells

Classification: Cell Culture.

Guidelines: Because neural stem cells (NSCs) have the potential of self-renewal and multidirectional differentiation, the method of suspended neural bulb culture can be used to obtain and study.

Cell Passage Protocol

Classification: Cell Culture.

Guidelines: Cell subculture is one of the conventional methods of cell culture. once the cells are overgrown in the flask, they need to be diluted and seeded into multiple vials before the cells can continue to grow. This process is called passage.

Isolation Protocol of Mouse Neural Stem Cells

Classification: Cell Isolation.

Guidelines: Neural stem cells (NSCS) are special cells with self-renewal and multidirectional differentiation potential. It not only can be used to explore the molecular mechanism of nervous system development, but also can be used as an alternative means for the treatment of central nervous system injury.

Flow Cytometry Staining Protocol of Mouse Neural Stem Cells

Classification: Cell Staining.

Guidelines: Cultured cells or tissue samples are made into single-cell suspension, and then cells are incubated in a flow tube or EP tube with fluorescence-labeled or unlabeled antibodies (It depends on the antibody). There are several ways of incubation, such as ice incubation, normal temperature incubation, and 37°C incubation.

Flow Cytometry and Collection Protocol of Mouse Neural Stem Cells

Classification: Flow Cytometry.

Guidelines: In a flow cytometry separation device, different cell subpopulations are labeled with different fluorescent antibodies and carry different physical (particle size, density, fluorescence intensity) information to separate target cells from the mixed cell population.

Primary and Passage Culture Protocol of Mouse Embryonic Fibroblasts

Classification: Cell Culture.

Guidelines: Mouse embryonic fibroblasts are suitable feeder cells for mouse or human embryonic stem cells and induced pluripotent stem cells. They can produce factors that inhibit the autonomous differentiation of embryonic stem cells and promote the proliferation of embryonic stem cells, which can effectively promote the proliferation of cells and maintain their undifferentiated characteristics and pluripotency.

Passage Culture Protocol of mESCs

Classification: Cell Culture.

Guidelines: The process of ESCs’ culture includes three parts of work, the culture of feeder cells, the preparation of feeder monolayers, and the culture of ESCs.

Resuscitation and Cryopreservation Protocol of mESCs

Classification: Cell Preservation.

Guidelines: If mESCs need to be frozen, it is best to do so at an early stage of culture. Select cells with normal cell morphology and karyotype, and store them in liquid nitrogen for a long time. The method is the same as that for freezing and resuscitation of general cell lines, while the freezing solution is based on the culture medium with 10%-15% DMSO.

Establishment and Culture Protocol of mESCs Cell Lines

Classification: Establishment and Culture Cell Lines.

Guidelines: Embryonic stem cells (ESCs) are abbreviated as ES, EK, or ESC cells. They are a class of cells isolated from early embryos (before the proto-intestinal stage) or primitive gonads, which have the properties of unlimited proliferation, self-renewal and multidirectional differentiation in vitro in culture. ES cells can be induced to differentiate into almost all cell types of the organism, both in vitro and in vivo environments.

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Immunology Protocols

Immunology is the study of the immune system and is a very important branch of the medical and biological sciences. The immune system protects us from infection through various lines of defense. Creative Bioarray provides an easily accessible reference for immunology techniques, including immunohistochemistry (IHC), immunofluorescence (IF), immunoprecipitation, etc. We provide step-by-step guides to techniques including guidelines, methods, and notes.

Immunohistochemistry (IHC-P) Protocol

Classification: IHC.

Guidelines: Immunohistochemistry (or IHC) is a method for demonstrating the presence and location of proteins in tissue sections. Though less sensitive quantitatively than immunoassays such as Western blotting or ELISA, it enables the observation of processes in the context of intact tissue. This is especially useful for assessing the progression and treatment of diseases such as cancer.

IHC Protocol for Free Floating Brain Sections

Classification: IHC.

Guidelines: Identifying multiple proteins within the same tissue allows for assessing protein colocalization, is cost effective, and maximizes efficiency. Here, we describe a protocol for multiplex immunolabeling of proteins in free-floating brain sections.

IHC Protocol for Mouse Tissue Sections

Classification: IHC.

Guidelines: Immunohistochemistry is the study of localization, characterization and quantification of antigens (peptides and proteins) in tissue cells by applying the basic principle of immunology antigen-antibody reaction, through chemical reactions to develop color of chromogenic agents that label antibodies.

IHC Protocol of SABC and SV Methods

Classification: IHC.

Guidelines: The Strept Avidin-Biotin Complex (SABC) method is a simple and sensitive immunohistochemical method for displaying antigen distribution in tissues and cells. The SV (Super Vision) two-step method uses a polymerization labeling method to link peroxidase with secondary antibodies to form a super-sized antibody-enzyme polymer, replacing secondary and tertiary antibodies in the traditional method.

Immunofluorescence (IF) Protocol

Classification: IF.

Guidelines: Immunofluorescence is a technique used for light microscopy with a fluorescence microscope and is used primarily on biological samples. This technique uses the specificity of antibodies to their antigen to target fluorescent dyes to specific biomolecule targets within a cell, and therefore allows visualization of the distribution of the target molecule through the sample.

IF Protocol for General Cells

Classification: IF.

Guidelines: Immunofluorescence (IF) staining labels a specific target antigen with a fluorescent dye such as fluorescein isothiocyanate or cyanine. The fluorescent stain visualized under a fluorescent microscope allows for an assessment of the target molecule/protein distribution in the sample. The following protocol provides a method of immunofluorescence of general cells.

IF Protocol for Cardiomyocytes

Classification: IF.

Guidelines: Immunofluorescent labeling is a straight-forward technique for assessing the presence and the subcellular localization of antigens and/or proteins. The following protocol provides a method describing a suggested protocol for sarcomeric actinin, ANP, ER and ERb fluorescent staining of cardiomyocytes.

IF Protocol for Frozen Sections

Classification: IF.

Guidelines: Immunofluorescence technique, also known as fluorescent antibody technique, is one of the earliest developments in labeled immunology techniques. Some scholars have tried to combine antibody molecules with some tracer substances to localize antigenic substances in tissues or cells using antigen-antibody reactions since a long time.

Simultaneous Double IF Protocol

Classification: IF.

Guidelines: In order to be able to examine the co-distribution of two (or more) different antigens in the same sample, a double immunofluorescence procedure can be carried out. Primary antibodies raised in different species can be used either in parallel (in a mixture) or in a sequential way. We provide a protocol for immunofluorescent double staining incubating the antibodies together.

Sequential Double IF Protocol

Classification: IF.

Guidelines: Double immunofluorescence is used to detect the presence and localization of two different proteins or antigens in a biological sample. This technique is commonly used in research areas such as immunology, cell biology, and pathology. We provide a protocol for immunofluorescent double staining incubating the antibodies separately.

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Molecular Biology Protocols

Molecular biology is the branch of biology that studies the molecular basis of biological activity. Molecular biologists conduct experiments to investigate the structure, function, processing, regulation, and evolution of biological molecules and their interactions with one another, providing micro-level insights into how life works. Creative Bioarray provides an easily accessible reference for molecular biology techniques. We provide a step-by-step guide to techniques including guidelines, methods, and notes.

In Situ Hybridization (ISH) Protocol

Classification: ISH.

Guidelines: In situ hybridization (ISH) is a type of hybridization that uses a labeled complementary DNA or RNA strand (i.e., probe) to localize a specific DNA or RNA sequence in a portion or section of tissue (In Situ) or in the entire tissue (whole mount ISH). Localization of endogenous transcripts is a desirable approach for confirming expression patterns.

ISH Protocol for Determination of TNF-mRNA

Classification: ISH.

Guidelines: Tumor Necrosis Factor (TNF) induces apoptosis in a variety of cancer cells playing a role in cancer growth and metastasis. In situ hybridization (ISH) is a technique that uses colorimetric or fluorescent probes to target and visualize specific DNA or RNA sequences within tissues. Here, we present the mRNA of TNF determined by in situ hybridization.

ISH Protocol for Whole-Mount Embryos

Classification: ISH.

Guidelines: The ability to visualize the expression of a gene in both time and space is an essential tool of developmental biology. Here, we detail a robust method for in situ hybridization of RNA probes to whole pieces of fixed tissue. This method has been optimized for reliable and sensitive visualization of the spatial patterns of gene expression in mouse embryo tissue.

ISH Protocol with Photosensitive Biotin Nucleic Acid Probes

Classification: ISH.

Guidelines: Photosensitive biotin has a linker arm with biotin attached at one end and an aryl azide compound at the other. Under visible light irradiation, the aryl azide compound may become activated aryl nitrobenzene, which readily binds specifically to the adenine N-7 position of DNA or RNA, binding approximately one biotin per 50 bases.

ISH Protocol with Nonradioactive Probes

Classification: ISH.

Guidelines: We describe a nonradioactive method for the detection of mRNAs. The biotinylated complementary oligonucleotide or cRNA probes hybridize with the cytoplasmic mRNAs and are detected by antibiotics. The reaction is amplified by a sandwich technique that provides layers of biotin and streptavidin-peroxidase.

Radioactive ISH Protocol for Animal Chromosomes

Classification: ISH.

Guidelines: Although largely replaced by the use of fluorescent in situ hybridization (FISH) in animal and human molecular cytogenetics, the technique of radioactive in situ hybridization (RISH) still has some uses. The sensitivity of RISH can be increased with longer exposures, in a roughly linear fashion until the silver bromide grains in the emulsion approach saturation over the target.

Radioactive ISH Protocol for Detecting Gene Expression Patterns

Classification: ISH.

Guidelines: Knowing the timing, level, cellular localization, and cell type that a gene is expressed in contributes to our understanding of the function of the gene. Each of these features can be accomplished with in situ hybridization to mRNAs within cells.

Nonradioactive ISH Protocol for Frozen Sections

Classification: ISH.

Guidelines: Nonradioactive in situ hybridization offers a unique opportunity to study gene expression on samples with preserved histological information. This method makes it possible to locate not only wherein a tissue a particular gene is expressed, but in many cases also in which specific cell type it is active.

ISH Protocol for Electron Microscopy

Classification: ISH.

Guidelines: In the great majority of cases in situ hybridization is used to localize mRNA species at the tissue level, or DNA at the chromosome level. These approaches are generally best done by light microscopy. There are instances, however, when it becomes important to localize nucleic acids at the subcellular level, this brings us into the domain of the electron microscope.

ISH Protocol to Polytene Chromosomes

Classification: ISH.

Guidelines: The utilization of in situ hybridization technology is of particular interest to those engaged in chromosome walking or genome mapping projects, in which it is essential to check all clones along a chromosome walk by in situ hybridization to identify clones containing repetitive DNA and to avoid the isolation of clones derived from regions outside that of interest.

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Molecular Biology Protocols

Genetics is the scientific study of genes and inheritance, certain qualities or characteristics are passed from parents to offspring due to changes in the DNA sequence. A gene is a segment of DNA that contains instructions for building one or more molecules that help the body work. creative Bioarray provides an easily accessible reference for genetic techniques including chromosome analysis, gene transfer, transgenic animal models, and more. We provide step-by-step technical guides, including guidelines, methods and instructions.

Chromosome Painting Protocol of Mouse Chromosomes

Classification: Chromosome Analysis.

Guidelines: Chromosome painting enables the visualization of chromosomes and has been used extensively in cytogenetics. Chromosome paint probes, which consist of a pooled composite of DNA-FISH probes, bind to nonrepetitive sequences for individual chromosomes.

Protocol for Chromosome Preparation in Human Cancer Cells

Classification: Chromosome Preparation.

Guidelines: Corresponding cell lines have now been established for most cancers. Cancer cells cultured in vitro mostly belong to the continuous cycle cells, which can be used to prepare chromosomes.

Protocol for the Preparation of Human Peripheral Blood Chromosomes

Classification: Chromosome Preparation.

Guidelines: Human peripheral blood small lymphocytes, which are usually in the G1 (or G0) phase, do not normally divide. If phytohemagglutinin (PHA) is added to the culture medium, such small lymphocytes can be stimulated to transform into lymphoblasts and enter mitosis. After short-term culture, a large number of mitotic cells can be obtained by colchicine treatment, hypotonicity, and fixation.

Chromosome G Banding Technology Protocol

Classification: Chromosome Analysis.

Guidelines: After treatment with physical and chemical factors, the chromosomes are then differentiated and stained with dyes, so that each chromosome appears in the light and dark, or different shades of banding technique is called banding technique.

iPSC Reprogramming Protocol Using Sendai Virus-Mediated Gene Transfer

Classification: iPSC Reprogramming.

Guidelines: Peripheral blood mononuclear cells (PBMCs) are cultured to expand the erythroblast (EB) population. They are then used to derive iPS cells using four recombinant Sendai viral vectors, expressing the four reprogramming factors Oct4, Sox2, Kfl4, and c-Myc.

Protocol for Making Retroviral Reprogramming Factors

Classification: Making Reprogramming Factors.

Guidelines: This protocol was developed as an efficient and consistent method to produce functional retroviral reprogramming factors. It avoids the need for concentrating virus via ultracentrifugation.

Nuclear Transplantation Protocol of Mammalian Cells

Classification: Nuclear Transplantation.

Guidelines: Nucleus transplantation, where the nucleus of one cell is put into another cell by microinjection. The former is the donor, which can be the stem cell nucleus of an embryo or the nucleus of a somatic cell. The recipient is mostly the egg of an animal.

Protocol of pcDNA3.1-GFP Transfection Cells

Classification: pcDNA3.1-GFP Transfection.

Guidelines: The liposome transfection method refers to the positively charged surface of cationic liposomes, which can be with the phosphate of nucleic acids through electrostatic interaction of DNA molecules wrapped into the formation of DNA a lipid complex.

Sequence and Ligase Independent Cloning (SLIC) Protocol

Classification: Cloning.

Guidelines: SLIC cloning is a cloning method based on recombinant DNA technology, in which gene fragments are spliced by using linearized segments of exogenous and target DNA to interface with each other and join them using sequence-independent ligases.

Blunt-End TOPO Cloning Protocol

Classification: Cloning.

Guidelines: TOPO cloning technology is based on the interaction of a special transposon sequence, the pUC18/19 vector, transposase, and DNA polymerase.

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