Immunhistochemstry
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| any substance that can induce a detectable immune response is | immogen or antigen
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| best antigens | proteins
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| polysaccharides, nucleic acids, other polymers | antigenic
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| most common antigens that induce antibody production by the body | bacteria and virus
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| commonly known as immunoglobulin | antibodies
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| Proteins that are produced by B lymphocytes in response to antigen stimulation | antibodies
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| IgG, IgA, IgM, IgD, IgE | Five classes of antibody
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| differ in structure and function but have the same basic structure | antibodies
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| IgG, IgA, IgM, IgD, IgE | composed of two identical heavy chains (H) and a two light chains (L)
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| Difficult to standardize because of variability of the immune response from different animals | polyclonal antibody productions
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| created from pools of many animals | polyclonal antibodies
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| allows for less variation from batch to batch | polyclonal antibodies
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| prepared by immunizing mice w/an antigen | monoclonal antibody
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| B lymphocytes are harvested from the mouse spleen | Monoclonal antibody
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| These lymphocytes are fused w/a nonsecreting myeloma cell (nonsecreting plasma cell tumor) | Monoclonal Antibody
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| This in vitro fusion yield hybrid cells (hybridoma) that retain the antibody secreting capibility of the B cells a/t immortality of the tumor cells | MAB
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| these cells can be cloned and a single cell us capable if producing an antibody identical to the originial | Hybridoma Cells
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| Can be characterized, standardized and produced in unlimited quantities | MAB
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| can be produced in other animals but it is usually done in mice | MAB
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| Advantages-high homogeneity, absence of nonspecific antibodies, no batch to batch variability | MAB
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| Are purer than PAB, display high affinity and selectivityof polyclonals w/o displaying most of the undesirable characteristics | MAB
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| immunohistochemical staining method, Direct, Indirect, unlabeled or soluble enzime immune complex, avidin biotin | various techniques used to detect the presence of antigen in pt's tissue or serum
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| labeled antibody of known origin is used to identify antigens in the patients tissue | Direct Method
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| The antibody may be labeled w/a fluorescein isothiocyanate,or fluorescent dye or w/an enzyme such as horseradish peroxidase, alkaline phosphatase or glucose for subsequent reaction w/a chromogen for light microscope evaluation | Direct Method
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| Patients serum is added to tissue sections containing known antigens to test the patient for the presence of antibodies to antigens | Indirect Method
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| antinuclear antibodies | Examples indirect method
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| has also been used to denote a method using two antibodies to detect antigen in a patient's tissue | Indirect method
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| is really a direct method | two step indirect
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| The first antibody is an unlabeled defined antibody and the second labeled is used to localize the first antibody | Two step indirect
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| Both the second and Third Antibodies are labeled w/enzymes | Three step method
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| increased staining intensity | Three step method
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| This is a three step method using primary antibody or secondary antibody and soluble enzyme-antienzyme complex | unlabeled or soluble enzyme immune complex method
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| The primary and the enzyme-antienzyme complexes must be made in the same animal species for the secondary antibody to link them together | unlabeled or soluble enzyme immune complex method
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| use either PAP or alkaline phosphatase antialkaline phosphatase immune complex | the most common techniques in unlabeled or soluble enzyme immune complex method
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| ABC - Avidin-biotincomplex, LAB - labeled avidin-biotin | there are two avidin-biotin techniques
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| Primary antibody is followed by a Biotinylated secondary antibody,The third step is the application of an avidin biotin enzyme complex | ABC
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| Antibodies from multiple clones/B Lymphocytes, bind to different epitopes obtained from an immunized animal | Polyclonal antibodies
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| Has low background, inexpensive and sensitive | ABC
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| The primary antibody is followed by a biotinylated secondary antibody. the third step, Application of an enzyme labeled avidin | Labeled avidion biotin (LSAB)
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| 4-8 times more sensitive than the ABC method | Labeled Avidin Biotin method
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| Immunoflouorescense, enzyme immunohistochemstry | two methods of visualization
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| antigens can be visualized in tissue sections as well as live cells | Immunofluorescence
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| a dye that absorbs light and then emits its own light at a longer wavelenghth | Flurochrome
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| when fluorchorme is attached or conjugated to the antibody, the sites of reaction between antigen and labeled antibody c/b seen | Flurochrome
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| can be detected by immunofluorescence | Any antigen
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| pathologys oldest immunohistochemical tool | immunofluorescense
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| sensitive, specific and simple makes this method very useful | immunofluorescense
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| Most commonly used fluorochromes in immunofluorescence techniques, both dyes absorb light that is not visible to the human eye and emit visible light | FITC and Rhodamine
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| Specimens examined w immunofluorescent techniques | kidney and skin bx
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| in presecence of a substrate and a chromogen, provides indicator system to visualize the location of the antibody | the enzyme
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| alkaline phosphatase, beta galactosidase, glucose oxidase and horseradish peroxidase | variety of enzymes used as markers
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| enzyme most commonly used for coupling to antibody | horseradish peroxidase and alkaline phosphatase
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| in the presence of hydrogen peroxide and a chromagen such as DAB or AEC will identify the sites of the antibody by forming a colored complex | horseradish peroxidase
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| demonstrated by napthol-AS-phosphate and a chromagen such as fast red-violet LB,fast red TR or fast blue BBN. all of the mentioned chromagens excpet DAB are soluble in organic solvents so aqueous mounting media must be used | Alkaline phosphatase
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| Mayers hematoxilyn is recommended, it doesnt contain alcohol | When hematoxilyn is used as a counterstain,
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| dissolves product and give a false negative result | when harris or another hematoxylin solution containing alcohol is used w/AEC or alkaline phosphatase chromogens
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| known as antigen retrieval | epitope enhancement or retrieval
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| compromised by some fixatives expecially aldehydes | immunoreactivity
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| causes excessive crosslinking of proteins | overfixation of tissues by formaldehyde
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| results in antibodies do not have access to their respective epitopes and false negative stains may result | Overfixation of tissue
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| HIER -heat induced retrieval and EIER Enzyme induced epitope retrieve | Two methods of unmasking or enhancing epitope
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| HIER -Original method used | Microwave
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| Microwave w/a pressure cooker, steamer, autoclave, water bath | HIER items used
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| provide the greatest epitope retrieval, effective and ease | microwave/pressure cooker
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| the oldest method of epitope retrieval | EIER
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| most common enzyme used | trypsin
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| Pronase, protease, ficin, pepsin are also used | Enzymes used in EIEr
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| digestion time for proteolytic enzymes | 1 to 60 minutes or more
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| reduces nonspecific staining but can increase it, also may weaken specific staining, create false negative results and cause fragmentation or loss of tissue sections | proteolytic enzyme digestion
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| ability to further dilute antibodies | advantage of antigen retrieval
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| exposure of epitope sites not previously detected | Advantage of antigen retrieval
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| more intense reactions w/ decreased incubation times | advantage of antigen retrieval
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| decreased background staining | advantage of antigen retrieval
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| more uniform staining, day to day consistancy of stains | advantage of antigen retrieval
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| possibility of better standardization | advantage of antigen retrieval
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| should be run along w/each antibody every time a run is performed | positive controls
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| commercially prepared slides can be used to check the reliability of the reagents, purchased slides should not be used as routine positive controls | positive controls
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| is one prepared by the lab under exactly the same conditions including type and timing of fixation and processing as the diagnostic slide | positive controls
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| run by substituting the primary antibody w/ nonimmune serum from the same species as the primary antibody or the diluent buffer used for the primary antibody | negative controls
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| control will not detect any nonspecific binding of animal serum components to the tissue and any staining observed w/b due to either endogenous peroxidase or binding of other antibody reagents | if diluent buffer is used
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| should be evaluated under the conditions: no retrieval, heat induced retrieval, enzyme induced retrieval, heat and enzmyme induced retrieval | New antibodies
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| optimal anitbody dilution must be determined, so use several different dilutions using the manufacturers recommendations | new antibodies
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| Hydrogen Peroxide, Nonimmune serum | two blocking reagents
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| first the use of hydrogen peroxide usually prepared in absolute methanol to block endogenous peroxidase activity (RBC's) | blocking reagents
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| occurs as a result of the antibody (protein) attachment to highly charged collagen and connetive tissue elements | nonspecific background staining
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| when protien applied to the tissue is the primary antibody. | nonspecific binding occurs
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| will bind to the nonspecific bound primary antibody and when reacted w/ the substrate-chromagen will give a positive result | secondary antibody
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| nonimmune serum from the species, the second antibody was produced in is | most commonly used
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| add an innocous protein to the tissue before the primary antibody. | prevents nonspecific binding
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| blot off blocking serum after incubating for 10-20 minutes and add the primary antibody | blot off
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| 10-20 minutes | incubation time
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| are cocktails of antibodies raised in different species, similar to universal link antibody | multilink secondary antibodies
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| mixtures of biotinylated anti-mouse, anti-rabbit and frequently other species | multilink secondary antibodies
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| allows you to stock less reagents | multilink secondary antibodies
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| primary antibody not addeded. substrate-chromagen improperly prepared | trouble shooting, specimen unstained positive control-unstained
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| reagents used in wrong order. alcohol based counterstain or mounting media used w/AEC, fast red or tetrazolium salts | trouble shooting, specimen unstained positive control-unstained
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| wrong secondary antibody used. omission of labeled reagent. sodium azide in the buffer baths | trouble shooting, specimen unstained positive control-unstained
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| no antigen in specimen, antigen masked during fixation | specimen-unstained, positive control-stained
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| Fixative too harsh for the antigen, antigen destroyed. | specimen unstained, positive control-stained
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| Use fixation recommended by antibody manufacturer on future specimens. | specimen unstained, positive control-stained
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| tissue exposed to too high heat. check oven temp | specimen unstained, positive control-stained
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| substrate-chromagen improperly prepared | specimen-weak staining, positive control-weak staining
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| primary anitbody too dilute or defective | specimen-weak staining, positive control-weak staining
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| insufficient incubation time | specimen-weak staining, positive control-weak staining
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| one or more defective reagents | specimen-weak staining, positive control-weak staining
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| too rinse buffer left on slides, diluting reagents excessively | specimen-weak staining, positive control-weak staining
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| antigen retrieval method done incorrectly | specimen-weak staining, positive control-weak staining
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| atingen present in low concentrations | specimen-weak staining, positive control-stained
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| antigen masked during fixation. try antigen retieval methods. | specimen-weak staining, positive control-stained
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| paraffin incompletely removed | specimen-excessive background, positive control-excessive background
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| many cells containing endogenous peroxidase | specimen-excessive background, positive control-excessive background
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| excessive adhessive used on slides. slides not washed well with buffer | specimen-excessive background, positive control-excessive background
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| concentration of primary antibody, secondary antibody or label reagent too high | specimen-excessive background, positive control-excessive background
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| incubation time of primary antibody, secondary antibody or label reagent too high | specimen-excessive background, positive control-excessive background
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| free antigen in tissue because of necrosis, autolysis, or degeneration. interpre in areas of less intense background staining | specimen-excessive background, positive control no background
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| Immunoglobin G (IgG), IgA, IgM, IgD and IgE. | each is composed of two identical heavy chains(H) and two identical light chains (L).
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| Differ in antigenic and structual properties, and determine the class and subclass of the molecule | the H chanis
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| are either of type kappa (k) or lambda | the two L chains
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| differs in all Ig classes and subclasses as well as between different species. | distribution of kappa and lambda
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| join L and H | covalent interchain disulfide bridges. by participatin in the tertiary structure, they confer greater stability to the immunoglobin molecule
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| If you draw blood from your arm and Igm antibodies were isolated and then injected into a rabbit your IgM antibody would act as an antigen and stimulate the rabbit to make antihuma IgM antibody. Serum from the immunized rabbit could then be used as a poly | Polyclonal antibody production
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| substance acted upon by an enzyme | substrate
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| colored compound can be converted to a dye | chromogen
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| patients serum may also be added to a known bacteria to detect the presence of bacterial antibodies in patient | Indirect method
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| usually used w/enzyme conjugated antiboides for light microscopy | two step and three step method
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| has a high affinity for vitamin biotin | avidin
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| the primary antibody is followed by a biotinylated secondary antibody (linking antibody). | in both the ABC and LAB method
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| is four to eight times more sensitive than the ABC method | LAB method
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| makes it possible to visualize antigens in tissue sections or live cells | Immunofluorescence
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| absorption and emission of light | fluorescence
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| use mayers hematoxilyn, if a hematoxilyn containing alcohol is used w/AEC or alkaline phosphatse chromogen, the product will disolve giving a false negative result | enzyme immunohistochemistry
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| detectability of many antigens in formalin fxd tissue is greatly improved by | epitope enhancement methods
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| ability to further dilute anitboidies, exposure of epitope sites not previously dected, more intense reactions with decreased incubation time, decreased background stain, day to day consistancy of stain, possibility of better standardization | advantages of epitope enhancement
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| binds to a primary antibody made in any of a variety of animal species or to both polyclonal and monoclonal antibodies | multilink secondary antibodies
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| localizes antigens | Basic PAP Immunoperoxidase procedure
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| principle- this method uses three reagents: primary antibody, a secondary antibody and a PAP comples that is composed of the enzyme peroxidase and an antibody against peroxidase | Basic PAP Immunoperoxidase procedure
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| The primary antibody is specific for the antigen. The secondary or "link" antibody is capable of binding to both primary antibody and to the PAP complex, both primary antibody and PAP complex are produced in same animal species | Basic PAP Immunoperoxidase procedure
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| fixation - b-5, zenker or bouin fxd tissue will not require epitope enhancement methods, some antigens aer not well demonstrated after fixation in these reagents. | Basic PAP Immunoperoxidase procedure
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| fixation - some formalin-fxd antigens may be better without the use of epitope enhancement methods, most antgens are masked by formalin fixation and the tissue will require some type of epitope retrieval | Basic PAP Immunoperoxidase procedure
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