Stack #212115

Description:	Immunhistochemstry
Category:	Histology
Created by:	nperez on 2009-02-25

Question	Answer
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	antibody
Proteins that are produced by B lymphocytes in response to antigen stimulation	antibody
IgG, IgA, IgM, IgD, IgE	Five classes of antibody
differ in structure an function but have the same basic structure	antibody
IgG, IgA, IgM, IgD, IgE	composed of two identical heavy chains (H) and a two light chains (L)
Diffucult 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 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 b cloned and are 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 selectivity	MAB
Direct, Indirect, unlabeled or soluble enzime immune complex, avidin biotin	immunohistochemical staining method
labeled antibody of known origin is used to identify antigens in the patients tissue	Direct Method
The antibody may be labeled w/a fluorescent dye or w/an enzyme such as horseradish peroxidase 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	the most common techniques
ABC - Avidin-biotincomplex, LAB - labeled avidin-biotin	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 followede 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
is attached or conjugated to the antibody	Flurochrome
can be detected by immunofluorescence	Any antigen
pathologys oldest immunohistochemical tool	immunofluorescense
sensitive, specific and simple	immunofluorescense
Most commonly used fluorochromes in immunofluorescence techniques	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 horsradish peroxidase	enzyme variety
enzyme most commonly used for coupling 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 but 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 corsslinking of proteins	overfixation of tissues by formaldehyde
results in antibodies not having 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 retrieval
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	multilink secondary antibodies
mixtures of biotinylated anti-mouse, anti-rabbit and frequently other species	secondary antibodies
allows you to stock less reagents	multilink secondary antibodies
primary control 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. use antigen retrieval	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