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UND 363 IHC
| Question | Answer |
|---|---|
| Define Primary Antibody | a primary antibody targets a specific antigen |
| Define Secondary Antibody | a secondary antibody is able to bind to the primary antibody which in turn is attached to the antigen |
| Define affinity | the strength of the attraction/binding to an antigen |
| Define sensitivity | sensitivity is the ability of a primary antibody to be diluted and still give a good stain |
| Define Specificity | specificity is the binding to a specific epitope and not to others |
| Define Chromagen | a chromagen is the colored reaction product or colored end product of an IHC stain |
| Define Flourochrome | a dye that will absorb light and then put out its on light of a longer wavelength |
| Define Epitope | it is the part of the antigen that is recognized by the antibody |
| DAB means | 3,3' diaminobenzidine |
| PAP means | peroxidase-anti-peroxidase |
| APAAP means | alkaline phoshpatase-anti-alkaline phosphotase |
| IHC means | Immunohistochemistry |
| EIER means | enzyme induced epitote retrieval |
| HIER means | heat induced epitote retrieval |
| ABC means | avidin-biotin enzyme complex |
| LAB means | labeled avidin biotin |
| FITC means | flourescein isothiocyanate |
| TRITC means | Tetramethylrhodamine isothiocyanate |
| DAPI means | 4',6-diamidino-2-phenylindole |
| Why use epitope retrieval methods? | epitope retrieval breaks down formalin fixation hydrogen bonds thereby allowing better detection of antigens. |
| Give two common flourochromes and the colors they produce | FITC - green, TRITC - red |
| Describe Monoclonal antibodies | monoclonal antibodies are produced by injecting subject with an antigen, the antigen reacted lymphocytes are fused with non secreting myeloma cells that create a hybrid cell that can be cloned individually |
| Monoclonal has what sensitivity, specificity, and background staining | monoclonal antibodies have a high specificity since they are cloned for one antigen and will have low background staining due to their specificity. Monoclonal antibodies have a low sensitivity. |
| Describe Polyclonal antibodies | subject injected with antigen (4 epitopes), subject produces 4 antibodies which then clone from b-lymphocytes, each clone can bind to a specific antigen. Which in turn creates a multitype antiserum. |
| Polyclonal antibodies have what sensitivity, specificity, and background staining | polyclonal is more sensitive since it can bind to more than one epitope but will also produce non-specific staining. There is low specificity since it binds to so many epitopes. |
| What are blocking reactions (in relation to immunoperoxidase method) | blocking reactions stop unwanted activity or staining. The first is the use of hydrogen peroxide to block endogenous peroxidase activity, the second is serum blocking via a nonimmune serum from the same host or animal. |
| what are the advantages of epitope retrieval | can further dilute antibodies, open previously closed epitope sites, give better/more intense reactions, decrease background staining and more consistency of that stain. |
| give advantages to monoclonal antibodies | low background staining, high specificity, no lot variability, and high homogeneity |
| give disadvantages to monoclonal antibodies | will have low antigenic sensitivity |
| what are the advantages of polyclonal antibodies | high sensitivity via binding of multiple epitopes of an antigen |
| what are the disadvantages of polyclonal antibodies | high background staining, low specificity, high lot variability |
| what is the advantage of immunoflourescence | immunoflourescence has high sensitivity and specificity |
| what is the disadvantages of immunoflourescence | high levels of background staining, requires frozen sections and a flouroscope, and will have poor resolution morphologically. |
| what are positive and negative controls (per IHC) | positive controls will always stain positive for the specific antigen, negative controls will not stain due the absence of the antigen. Positive controls will validate the stain is working and the antigen is present/viable. |
| what are the classes of immunoglobins and which is the most common? | igG, igA, igM, igD, igE, with immunoglobin g (igG), being the most common. |
| Why use immunohistochemical stains? | They are diagnostic techniques that aid the pathologist in making a determination of patient care specifically related to tumors and their treatment. |
| describe the direct IHC staining method | direct - antibodies are "labeled"/bound with an enzyme, flourochrome or colloidal gold and attached directly to epitope then visualized with a microscope. Enzymes require a reaction to produce a color which can then be seen by a light microscope |
| describe the indirect IHC staining method | an "unlabeled" primary antibody binds to the antigen. A secondary antibody is linked to the primary and has a "label" so that it can be seen by the labels detection method. |
| what are the differences between direct and indirect IHC stains | Direct method will use multiple labeling techniques, whereas indirect methods typically only use immunoflourescence techniques. |
| what are the most common epitote retrieval methods | HIER - heat induced epitope retrieval, and EIER - enzyme induced epitope retrieval. |
| define antigen | a molecule that can produce an immune response (protein, carbohydrate or other polymer) |
| What does Fab, Fv, Fc, and CDR's mean in realation to an antibody diagram | Fab - fragment antibody binding, Fv - Fragment variable, Fc - Fragment Crystallisation, CDR - complementarity determining region |
| explain the location of Fab, Fc, Fv, CDR in relation to the antibody diagram | Fab - are the full length parallel upper angled arms, Fc - is the stem (below the point of angle), Fv is the last half of the outer armS, CDR - is the last half of both parallel arms |
| What are the variable regions, constant region, light chain and heavy chain of the antibody diagram | the variable regions are the upper half of the parallel arms, the constant regions are everything below the variable regions, the light chain is the outer arm, the constant region is the inner arm and stem |
| what attaches the heavy chains and light chains | disulfide bonds |
| name three flourchromes and their colors | FITC, TRITC, and DAPI FITC is green, TRITC is red, and DAPI is blue |
| name 2 chromagens and their color they produce | DAB - Brown, AEC - Brick red |
| what are three excellent antigens | proteins, bacteris and viruses |
| what does the acronym GAMED mean in relation to immunoglobin classes | they are the 5 classes of immunoglobins, with IgG being the most common |
| what does and enzyme do | protein that acts as a catylist. |
| what does a catylyst do | compound that accelerates a chemical reaction |
| what is a chromagen | it is a colored reaction product (2 most common are DAB and AEC |
| how does the antibody as an antigen formulation work | a secondary antibody links to the primary antibody (that is attached to the epitope) via the Fc (stem) region |
| what is the general IHC staining Scheme (8) | epitope retrieval, block (usually hydrogen peroxide), block (serum block), primary antibody, link (secondary antibody), label (binds to link and give site for chromagen), chromagen (DAB/AEC), counterstain (Mayers, methly green, NFR) |
| What will underfixation and overfixation cause in regards to IHC problems | UNDER - crosslinking at periphery with deeper tissue "fixed" by alcohol, OVER - increased crosslinking and can cause false negatives |
| how will decalcification affect IHC | does not seem to interfere if tissues are well fixed with formaldehyde. BUT bone marrow bxs can be affected by harsh DC's such as nitric acid for extended periods |
| What methods can be used with HIER | microwave, pressure cooker, steamer, autoclave |
| What does a positive control do | tissue selected for IHC will always stain positive for antigen, validates that the antigen is present and viable and that the reagents worked. Must try and keeps all process identical in lab to limit variables |
| what can sausage blocks and micro arrays do for positive controls | they use multiple samples in a row to decrease the amount of slides necessary for a wide-range positive control |
| what is used for blocking background staining during IHC | hydrogen peroxide for endogenous peroxidase, a NON-IMMUNE serum for serum blocking (must be the host of the SECONDARY IgG) - others include calf serum, nonfat dry milk, casein, |
| What are the 5 methods of IHC staining | Direct, indirect, unlabeled enzyme comples, avidin biotin, traminase or CSA method, polymer method |
| how are direct flourochrome and enzyme labeled antibody stains seen | flourochromes are bound and seen with flourescent microscope, the enzyme reacts with a chromagen and then seen with light microscope |
| what is used in indirect IHC staining | a secondary antibody (species b) that is attached to a primary (species a). the secondary antibody is labeled; commonly with enzyme-anti-enzyme or peroxidase-anti-peroxidase. Chromagen reacts to the EAE or PAP to be visualized |
| What can the indirect staining with an enzyme have an advantage of doing | it can perform double staining. primary (at epitope) is bound to secondary which is bound to a stable immune complex (this "third" antibody provides the to variable site arms for the double staining. |
| what are the two methods of avidin biotin | ABC (avidin biotin complex) and LAB (labled avidin biotin) |
| what is the procedure for ABC and LAB | ABC - primary, biotinyliated secondary, avidin biotin complex, chromagen LAB - primary, biotinylated secondary, enzyme labeled avidin, chromagen |
| what are the advantages for ABC and LAB | high sensitivity (ABC 40x over other IHC; LAB 4x - 8x over others), low background, can use at higher dilutions |
| explain the tyramine amplification/CSA method | peroxidase enzyme catalyzes deposition of additional tyramine in a halo effect thereby creating a larger colored reaction product. is difficult to control and creates more background staining. |
| explain a polymer label | peroxidase molecules attached via a dextran chain (to antibody). This ensures no endogenous biotin and high sensitivity. |
| what does cish and fish stand for | chromagenic in situ hybridization, flourescent in sity hybridization. |
| explain the in situ hybridization | probe attached to tissue section, tissue dna strands are denatures (split into two) and then the single strands recombine with the "probe" strands |
| what are the 5 heavy chains and 2 light chains | GAMED, and lambda and Kappa |
| what other fixatives are good for antigen preservation | zenker, bouins, methanol-carnoy solution. |
| what will glutaraldehyde do to antigens | preserves morphology but irreversably blocks antigenic determinants |
| What will occur to the AEC chromagen if introduced to alcohol | will decolorize immediately. |
| what are the sections cut at for basic pap immunoperoxidase, ABC immu'dase, and HRP "horseradish peroxidase" | 4-5 for basic and 3-4 for the other two. |
| what type of slides should be used with the three IHC stains | positively charged |
| if the specimin and pos. control are unstained what are the likely causes | primary antibody not added, substrate-chromage improperly prepared, reagents in wrong order, alcohol used with AEC/fast red, tissue not allowed to dry, wrong secondary antibbody, omission of labeled reagent, sodium azide in buffer |
| if spec and pos control are both unstained and the issue is the secondary antiody why would that be | possibly antirabbit secondary used with monoclonal primary |
| if there is weak staining of spec and positive control what are the likely issues | substrate-chromage improperly prepared, primary antibody too dilute or defective, insufficient incubation, 1 or more defective reagents, too much rinse buffer on slides, epitope enhancement incorrectly done |
| if spec has a weak stain and the positive control has a normal stain what are the issues | antigen is in low concentrations or masked during fixation. **repeat and use epitope retrieval procedure or overnight incubation with primary antibody** |
| if the spec and pos. control have excesive background stain what are the possible issues | paraffin removal not complete, many cells with endogenous peroxidase, excessive adhesive, slides not well washed with buffer, % of primary antibody/link/label too high, incubation too long |
| if stain on spec is excessive and there is none on the pos. control what is the issue | there is free antigen in the tissue due to necrosis, autolysis, or degeneration. |
| what can be used so that AEC can be mounted with a synthetic resin | crystal mount |
Created by:
mustangvxd
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