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Stack #212115

Immunhistochemstry

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