Staining Flashcards
What is the Direct staining method?
A labeled Ab of known specificity is used to identify Ag in patient tissue.
What is the Indirect staining method?
The patient’s serum is added to tissue sections containing known Ag to test the patient for the presence of Ab to those Ag. The patient’s serum can also be added to bacteria.
What is the Unlabeled/ Soluble Enzyme Immune Complex method?
A 3 step method involving a primary Ab, secondary Ab and a soluble enzyme-antienzyme complex.
What is the Avidin-biotin complex (ABC) method?
The primary Ab is followed by a secondary biotinylated Ab which bond irreversibly to Avidin and forms a complex.
What are three main benefits of the ABC method?
- There is low background staining
- Sensitivity can be up to 40X other immunoperoxidase methods
- Ab may be used at higher dilutions than in other methods.
What is polymeric detection?
The “gold standard” of IHC staining in which either a single strand (monomeric) or multiple strand (polymeric) molecules are fused with the secondary Ab which eliminates the need for ABC or patient serum. We did this in Vancouver.
Which detection system works better: monomeric or polymeric?
Monomeric, because there is increased sensitivity and Ab binding site penetration with single strand molecules. Turnaround time is also decreased.
What is the general procedure for polymeric detection?
Ab-polymer-chromogen.
What neoplasm does BCL2 test for?
Follicular lymphoma.
What neoplasm does CD117 test for?
Gastrointestinal stromal tumor.
What neoplasm does CD15 test for?
Hodgkin’s lymphoma.
What neoplasm does CD20 test for?
B cell lymphoma.
What neoplasm does CD3 test for?
T cell lymphoma.
What neoplasm does CK20 test for?
Colon carcinoma.
What neoplasm does Cytokeratin (Pankeratin) test for?
Carcinoma in general.
What neoplasm does Desmin test for?
Leiomyoma (smooth muscle tumor, common in uterus).
What neoplasm does ER test for?
Breast carcinoma.
What neoplasm does Glial Fibrillary Acidic Protein (GFAP) test for?
Glioblastoma multiforme.
What neoplasm does Gross Cystic Disease Fluid Protein (GCDFP-15) test for?
Breast ductal carcinoma.
What neoplasm does HER2 test for?
Breast carcinoma.
What neoplasm does HMB-45 test for?
Melanoma.
What neoplasm does CD45 test for?
Lymphoma.
What is another term for CD45?
Leukocyte common Ag.
What neoplasm does Melan-A test for?
Melanoma.
What neoplasm does PR test for?
Breast ductal carcinoma.
What neoplasm does S100 test for?
Schwannoma.
What neoplasm does Thyroid Transcription Factor (TTF1) test for?
Lung adenocarcinoma.
What neoplasm does Vimentin test for?
Seminoma or overfixation of tissue.
T/F: Precut control slides may be stored at room temperature indefinitely.
False, storing them at room temp is not as good as in the fridge.
T/F: The procedure for staining cytology smears is the same as for routine slides.
False, each different type of slide must have its own protocol on the stainer.
In the bloody areas of a tissue section stained with the immunoperoxidase technique, there is a marked reaction with RBCs. What happened?
There was a failure to use hydrogen peroxide, it is crucial if the tissue contains many RBCs.
Paraffin sections stained with the immunoperoxidase technique show excessive background staining. What happened?
The nonimmune serum was not applied. If the first protein solution applied to the tissue is the primary Ab, nonspecific binding can occur.
The skin control section for S100 was stained with the immunoalkaline phosphatase technique using fast red TR as the chromogen. It shows negative staining. What happened?
The sections were accidentally dehydrated and cleared and caused the chromogen to break down.
What is “Quenching”?
The blocking of naturally occurring excess endogenous enzyme found within a cell prior to IHC staining. Ex. in RBCs causing excessive background staining.
When is “Quenching” performed?
At the beginning of the procedure, after deparaffinization and heat is applied for most Ag.
What is the most notable exception to the “usual” Quenching protocol?
CD34, because it will be dissolved by the hydrogen peroxide during quenching so it needs to be applied in between the primary and secondary Ab.
What is the purpose of a primary Ab?
It is a large protein (Ab) that binds to other proteins in the patient tissue (Ag).
What is the purpose of a secondary Ab?
Secondary Ab bind to the primary Ab and become conjugated into a complex such as the ABC.
What are the two different detection systems?
Biotin-(Strept)avidin and polymer.
What is a flurophore?
Molecules that “glow” upon excitation with ultraviolet light.
What is an enzyme?
Molecules that serve to catalyze certain reactions. In this case, they convert a soluble compound into an insoluble “deposited” chromogen.
What is a substrate?
Molecules that react with the enzyme in order to “activate” it.
What is a chromogen?
Substances that get “caught in the crossfire” of the enzyme-substrate reaction and are deposited at the Ag site.
What fixatives may be used for IHC?
10% Neutral-buffered formalin, Bouin’s, B-5, and Zenker’s fixatives as well as 95% for cytology specimens.
How does fixation affect immunoreactivity?
The molecular conformation of most protein antigens is modified in such a way that that antibodies may not be able to accurately recognize the targeted epitopes. This modification is generally referred to as ‘cross-linking’ which results in what is commonly referred to as a ‘masking’ or ‘cloaking’ of epitopes*.
What is the effect of decalcification on immunoreactivity?
Many protein epitopes are retained and can be successfully localized by IHC.
What are the three types of decalcification?
- “‘Strong’ mineral/inorganic acids (such as Hydrochloric, Nitric
and Chromic) - Weak’ organic acids (such as Formic, Acetic and Picric); and
- Chelating agents (such as EDTA).
What are commonly employed reagents for Quenching peroxidase activity?
- Solutions of hydrogen peroxide in DI water or phosphate- or TRIS-buffered saline (PBS/TBS);
- Solutions of hydrogen peroxide in methanol;
- Commercially prepared (‘proprietary’) solutions such as UltraBlock™, **Peroxidazed 1™, **DEEB, and **BloxAll
What are commonly employed reagents for Quenching Alkaline phosphatase activity?
- Solutions of Levamisole in DI water;
- Commercially prepared (‘proprietary’) solutions such as DEEB™ or BloxAll™
How are slides that are to be stained with reagents that employ (horseradish-) peroxidase as the ‘label’ pre treated?
are incubated with a weak (i.e. 0.5 to 2.0%) solution of hydrogen peroxide; many labs simply immerse slides in 3% ‘household-grade’ peroxide.
What is the scientific basis for “protein blocking”?
To inhibit (non-specific) binding of charged elements to antibody reagents, potentially resulting in non specific staining (background staining).
What are some commonly used protein blockers?
- Powered bovine milk and/or powered albumin;
- Denatured bovine serum albumin (BSA);
- Purified or ‘synthetic’ casein (milk protein);
- Solution of denatured mouse, rabbit, sheep and/or goat serum;
What neoplasm does Podoplanin (D2-40) test for?
Podoplanin is selectively expressed in normal and malignant lymphatic endothelium, including Kaposi’s sarcomas, epithelioid mesotheliomas and seminomas.
What neoplasm does HBME-1 test for?
Hector Battifora mesothelial-1 (HBME-1) labels thyroid papillary carcinoma and follicular carcinoma but not normal thyroid making it a valuable marker for distinguishing thyroid malignancies from benign thyroid lesions.
What neoplasm does Calretinin test for?
Anti-calretinin labels mesothelial and Leydig cells under normal and neoplastic conditions and has been shown to be useful in differentiating mesothelioma from adenocarcinomas of the lung. Control is usually mesothelioma.
What neoplasm does CK5/6 test for?
Mesothelioma and lung squamous cell carcinoma but can also be tested on kidney, skin and esophagus.
What does Napsin test for?
Lung adenocarcinoma. The polyclonal Napsin A antibody exhibits cross reactivity with a number of non-pulmonary carcinomas such as colorectal carcinoma and renal cell carcinoma.
What does TTF-1 test for?
Thyroid transcription factor-1 (TTF-1) is a transcription factor that plays a role in regulating genes expressed within the thyroid, lung, and brain. Anti-TTF-1 immunohistochemistry is useful for identifying carcinomas of thyroid and lung origin and the control type is typically lung adenocarcinoma.
The peroxidase staining procedure in which reagents are linked exclusively by antigen-antibody reactions without any conjugation steps is:
Peroxidase-anti-peroxidase
The three critical parameters of the heat-mediated antigen retrieval procedure are:
Proper temperature, incubation time, and pH
A member of a genetically identical group of molecules or organisms is known as a/an:
Clone.
What is an enhancer?
In in-situ hybridization, an enhancer is a DNA-binding protein that influences the translation of genetic sequences.
What is an exon?
An exon is a coding sequence of a gene.
What is a gene?
A gene is a segment of DNA involved in the production of a specific product, usually a protein.
What does A single plasma cell (activated B-cell) produce?
Antibodies specific to a single antigenic determinant
The specific substrate of horseradish peroxidase is:
Hydrogen peroxide.
Describe the immunoperoxidase technique:
All immunoperoxidase staining techniques use the enzymatic activity of horseradish peroxidase, with its specific substrate, hydrogen peroxide. Horseradish peroxidase catalyzes the release of oxygen from hydrogen peroxide. When this reaction is combined with the oxidation of a chromogenic substrate, such as amino-ethyl-carbazol or diaminobenzidine, a colored polymer is formed that is visible by light microscopy.
The ability of an antibody to recognize and bind with a specific protein antigen is due to its:
Dimensional structure. An antibody’s ability to recognize and bind with a specific protein antigen is due to its three-dimensional structure, which fits the shape of the antigen.
What determines the dimensional structure of a Ag?
The three-dimensional structure can be determined by the linear amino acid sequence of the antigen epitope, or the conformational shape of the molecule.
What is Ab diversity?
Antibody diversity is due to different amino acid sequences of the variable regions of both heavy and light chains. Antibody diversity is based on differences in amino acid sequences which determine three-dimensional structure in the variable regions of both heavy and light chains.
When do Fluorochromes fluoresce?
They are illuminated by a specific wavelength of light. Each fluorochrome will fluoresce only when illuminated by a specific wavelength of light. The specific energy of the illumination knocks an electron of the fluorochrome atom to a higher orbit and when it drops back down to its natural orbit it emits a photon of a specific wavelength of lower energy, which we see as a color. FITC, for example, requires a wavelength of 490 nm, which is blue. It emits a lower energy, longer wavelength of 520 nm, which is green.
Heat induced epitope retrieval (HEIR) may be used for:
A. all tissue sections, for any antibody
B. all formalin-fixed tissue sections
C. those antibodies for which it has been determined to be optimal
D. frozen sections
Those antibodies for which it has been determined to be optimal.
Fluorochromes are available in several colors. In order to see these colors, a fluorescence microscope must be equipped with:
The correct wavelength filters. In order to see specific colored fluorochromes, a fluorescence microscope must be equipped with special filters that provide the proper excitation wavelength of light as well as the proper filters to see the light wavelengths produced.
When selecting reagents for peroxidase-anti-peroxidase (PAP) staining, the PAP complex should be prepared in the same (or closely related) animal species as the:
Primary Ab.
How is the secondary Ab used in PAP staining?
In PAP staining, the secondary antibody acts as a bridge between primary antibody and the PAP complex, which is raised in the same (or closely related) animal species as the primary antibody. For example, if the primary antibody is rat anti-x, the secondary is made in another animal directed against the first animal, such as goat anti-rat.
What are the four main advantages of fluorescent staining?
- It’s quick and works well in multiplex staining.
- It has non-masking qualities
- Sensitivity
- Labels have a small molecular footprint which makes them easy to conjugate with an Ab.
What are the four main disadvantages of fluorescent staining?
- They fade over time
- It is difficult to view cell morphology
- It requires viewing through a specialized microscope.
- Each fluorophore used needs a specific filter.
Which part of the primary Ab does the secondary Ab recognize?
The Fc (constant) portion.
What are the three HRP chromogens?
- Diaminobenzidine (DAB)- Brown
- Aminoethylcarbazole (AEC)- Red
- Tetramethylbenzidine (TMB)- Green
What are the three AP chromogens?
- Fast red- fuchsia
- Fuchsin- fuchsia
- BCIP-NBT-Blue
What are the three main advantages of using enzymatic labels?
- Permanent
- Using light microscopy makes it easier to see morphology
- You only need a light microscope
What are the three main disadvantages of using an enzymatic label?
- It takes more time than fluorescent techniques
- Its harder to interpret multiple stains due to masking
- Some chromogens are very toxic (DAB)
What is nuclear staining?
The nucleus of the cell is stained by the chromogen. Ex. ER, PR, PAX5, PAX8, SOX10
What is membrane staining?
The nuclear membrane is stained by the chromogen. Ex. many CD markers
What is cytoplasmic staining?
The cytoplasm of the cell is stained by the chromogen. Ex. PSA for prostatic adenocarcinoma.
What is a serial dilution?
A titration series in which each dilution is exactly half as strong as the prior.
How does water quality affect IHC staining?
Since both IHC ‘wash’ buffers and retrieval solutions are usually prepared using (DI) distilled/deionized water, it is imperative that it be of the highest quality; ‘poor’ water quality results in incorrect/variable pH values, which may have a significant effect on the binding affinity of primary and secondary antibodies.
How does Ab dilution affect staining?
‘Primary’ antibodies that are obtained as concentrates must be stored, prepared and handled properly in order to ensure desired reactivity; concentrates should always be diluted with an appropriate diluent, which is often specified on the datasheet provided by the vendor – use of DI water or PBS/TBS wash buffer as an antibody diluent is not recommended.
How do pretreatment procedures affect staining?
it is imperative that such reagents be prepared correctly and used under appropriate conditions; in addition, although the datasheet for a particular antibody may specify use of a citrate-based retrieval solution, for a number of reasons, it may be necessary to pretreat specimens in an EDTA- or Tris- based solution in order to obtain acceptable staining results.
How does positive control material affect IHC staining?
It is the only way to properly ‘validate’ a procedure; due to normal, biologic variability in the expression of every ‘marker’, the ideal control specimen (at least for use during validation procedures), will contain three (3) pieces of tissue (or cultured cells) that express the ‘target’ protein in varying amounts – the resulting slides should, therefore, have staining intensities of 1+, 2+, and 3+/4+.
What is considered a “mild” Ag retreival?
20 minutes in a citrate buffer and a lab grade waterbath set at 85C.
What is considered a “moderate” Ag retreival?
30 minutes in a Tris buffer and a vegetable steamer set at 95C.
What is considered a “harsh” Ag retreival?
10 minutes in an EDTA buffer and a programmable pressure cooker set at 120C. It should be noted that this method is not recommended.
What is a lyophilized Ab?
A ‘freeze-dried`- Ab that needs to be reconstituted with DI water.
What is the staining pattern in this image?
