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  • HE Stain
    Hematoxylin and eosin stain (HE stain) is one of the widely used stains in histology. This stain produces colors different tissue structures, which would otherwise be transparent, so that you can get a detailed view of the tissue. As its name suggests, H&E stain makes use of a combination of two dyes: haematoxylin and eosin. Haematoxylin can be considered as a basic dye (general formula for basic dyes is:dye+ Cl-) which is dark blue or violet, it binds to basophilic substances (such DNA/RNA - which are acidic and negatively charged); and Eosin is a red or pink stain that is Acidic / Negative. It binds to acidophilic substances such as positively charged amino acid side chains (e.g. lysine, arginine).
  • Fluorescent Protein Transfection
    The Fluorescent Protein contained vectors can be used as transfection markers and to label living cells. They do not contain an MCS. In these vectors, the fluorescent protein is constitutively expressed and can be detected by fluorescence microscopy or flow cytometry, providing direct visual evidence of transfection/cotransfection or the ability to monitor cells after transplantation experiments in vivo. Transfected cells can also be used in assays that require fluorescent labeling of whole cells. The most widely used Fluorescent protein is Green Fluorescent Protein (GFP), and Red Fluorescent Protein (RFP).  GFP and RFP are both versatile biological markers for monitoring physiological processes, visualizing protein localization, and detecting transgenic expression in vivo, GFP can be excited by the 488 nm laser line and is optimally detected at 510 nm, while RFP can be excited by the 488 nm or 532 nm laser line and is optimally detected at 588 nm.
  • Immunohistochemistry Test
    Immunohistochemistry (IHC) is a technique used to determine the presence and level of specific cellular proteins. IHC measures protein expression using specially labeled antibodies that can bind to the proteins of interest. The antibody is mixed with the cellular components of the tumor. After a set amount of time, the mixture is rinsed and only those antibodies attached to their protein targets will remain. The presence of the antibodies can be detected by viewing the sample under a microscope because areas containing bound antibodies will appear a different color than areas lacking antibodies. Samples with more protein will bind more antibody and therefore appear darker. This allows the test to reveal not only whether a protein is present but also the relative amount of the protein. Test results are based on the strength of the staining and the percent of cells stained. Procedures: 1. Tissue Fixation 2. Antigen Retrieval 3. Staining/Add primary antibody 4. Add secondary antibody 5. Add chromogen 6. Counterstain 7. Detection
  • Immunofluorescence Technique
    Immunofluorescence (IF) or cell imaging techniques rely on the use of antibodies to label a specific target antigen with a fluorescent dye (also called fluorophores or fluorochromes) such as fluorescein isothiocyanate (FITC) to specific biomolecule targets within a cell, and therefore allows visualization of the distribution of the target molecule through the sample. IF allows researchers to evaluate whether cells in a particular sample express the antigen in question. In cases where an immunopositive signal is found, immunofluorescence also allows researchers to determine which subcellular compartments are expressing the antigen. IF can be used on cultured cell lines, tissue sections, or individual cells. There are two different immunofluorescence assays: indirect immunofluorescence assay and direct immunofluorescence assay. For indirect immunofluorescence assay, the protocol mainly include tissue or cell treparation, tissue or cell fixation, serum blocking, primary antibody incubation, marked second antibody incubation, staining, result judgment and imaging. For direct immunofluorescence assay, there are only marked primary antibody been incubated without second ant...
  • In Situ Hybridization
    In situ hybridization (ISH) is a type of hybridization that uses a labeled complementary DNA, RNA or modified nucleic acids strand which allows for precise localization of a specific segment of nucleic acid within a histologic section. The underlying basis of ISH is that nucleic acids, if preserved adequately within a histologic specimen, can be detected through the application of a complementary strand of nucleic acid to which a reporter molecule is attached. Visualization of the reporter molecule allows to localize DNA or RNA sequences in a heterogeneous cell populations including tissue samples and environmental samples, Riboprobes also allow to localize and assess degree of gene expression.  There are four types of ISH probes: Double-stranded DNA (dsDNA) probes, Single-stranded DNA (ssDNA) probes, RNA probes (riboprobes), and Synthetic oligonucleotides (PNA, LNA). The probes can be labeled both by radioactive isotopes (e.g. 32P, 35S, 3H) and non-radioactive labels (e.g. biotin, digoxigenin, fluorescent dye (FISH)). FISH (Fluorescence In Situ Hybridization) is a method which based on the in situ hybridization techniques and consist in ...
  • Chemiluminescence technology
    Chemiluminescence is the emission of cold light as a result of a chemical reaction. When a chemical reaction occurs, the reactions either give off (exothermic) or absorb (endothermic) heat, but there are few kinds of chemical reactions in which the energy produced is given off not as heat but as light, that is chemiluminescence. Chemiluminescent Assay is an important technique in molecular biology. There are two ways in which chemiluminescent assay used in the lab: the one-step sandwich technique and the two-step immunocapture technique. The procedures are like below:
  • Immunocytochemistry (ICC)
    Immunocytochemistry (ICC) is a common laboratory assay that can confirm the expression and location of target peptides or protein antigens in the cell via specific combination of antibodies and target molecules, It offers a semi-quantitative means of analyzing the relative abundance, conformation and subcellular localization of target antigens. There are many methods to obtain immunological detection on tissues, traditional ICC techniques use chromogenic detection in which enzyme conjugated antibodies convert chromogen substrates to a colored precipitate at the reaction site, immunofluorescent labels are also used to show the reaction site. The cellular antigen was visualized using either fluorochrome-conjugated primary antibodies (direct detection) or a two-step method (indirect detection) involving an unlabeled primary antibody followed by a fluorochrome-conjugated secondary antibody.  The ICC protocol including fixation, permeabilization, blocking, immunolabeling, counterstaining and microscopic imaging. The process is like below: 1. Sample preparation: 2. Immunostaining: 3...
  • Co-Immunoprecipitation Assay
    Co-Immunopericpitation (Co-IP) is one of the most straightforward technique to study protein-protein interactions in vivo thereby elucidating signaling pathways. Same to Immunoprecipitation assay, Co-IP method also involves incubating specific antibody along with the protein of interest to form an immune complex. This complex is then precipitated onto an immobilized support, ex: antibody-coupled beads, and then wash to remove the unbound protein. Protein components in the complexes are then visualized by Western blot and analyzed by SDS-PAGE. However, there are some difference between Co-IP and IP, co-immunopericpitation is more focused on the additional molecules that are bound to the target protein by inherent interactions in the sample complex. Process: 1. Design an expression plasmid which contained target gene. 2. Transfection cells. 3. Split cells and incubate with the specific antibody which against the target protein in cell lysate. 4. The complex is immobilized onto agarose resin through protein A or G. 5. Analyse samples by SDS-PAGE and Western blot.
  • ELISA/Elispot Assay
    The enzyme-linked immunosorbent assay (ELISA) is a plate-based immunological assay designed for detecting and quantifying substances such as peptides, proteins, antibodies and hormones in biological samples. There are three different types of ELISA: direct ELISA, competitive ELISA and sandwich EILSA. Fig.1 Different types of ELISA Among the three types, sandwich ELISA is most widely used method for the detection of samples. Sandwich EILSA used two sets of antibodies for the product detection, the procedure is as follows: 1. The primary antibody (capture antibody) is the antibody which raised against the antigen of interest, they were coated on the EILSA plate before the sample was added, any excess and unbound antibody is then washed from the plate. 2. The sample was added into the plate, any antigen found in the sample would bind to the primary antibody which already coated on the plate, again, any excess sample is washed from the plate. 3. The secondary antibody (detection antibody) was added. The secondary antibody is labeled with an enzyme, and would binds to any target antigen already bound to the plate. 4. The...
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