DETECTION SYSTEMS Flashcards
What is photobleaching?
A fluorophore loses its ability to fluorescence due to the buildup of reactive oxygen particles due to its natural activity.
How can photobleaching be reduced?
1) limiting the amount of free oxygen radicals in the environment
2) decreasing the excitation light in intensity and duration
3) Using a low concentration of fluorochrome with high-quantum efficiency
What are some common applications of IF in pathology?
- FISH to detect gene aberrations in cells.
- The detection of single monolayer organisms such as bacteria and parasites.
- The visualization of cell structures by super resolution microscopy.
Define ISH
In-situ hybridization
Technique used to identify gene amplifications, deletions and translocations as well as chromosomal copy number changes.
What are the three different ISH techniques?
FISH (fluorescent)
CISH (chromogens)
SISH (silver)
Named for the system that is used to identify the probe
Why is CISH used more commonly than FISH?
- The type of microscope (brightfield) used for CISH is more readily available than the microscope used for FISH (fluorescence).
- Brightfield CISH allows for better visualization of tissue structures.
- CISH signals do not fade overtime like FISH.
- Documentation by visual acquisition is simpler with brightfield than with fluorescent scopes decreasing the evaluation time for CISH vs FISH.
What are the two major categories of fixation?
Chemical
Drying
What are the two most commonly used fluorochromes in IF?
FITC
Rhodamine
What are the two most common enzymes used for antibody visualization?
Horseradish phosphatase (HRP)
Alkaline phosphatase (AP)
How are DAB and AEC commonly used?
Visualize the sites of antibody binding by forming a colored compound
In Avidin-biotin methods, what follows the primary antibody?
The biotinylated secondary antibody
How does alkaline phosphatase function in some IHC methods?
As the enzyme
Define fluorochrome
A dye that absorbs light then emits its own light at a longer wavelength
How is horseradish peroxidase used in some Avidin-biotin methods?
As the enzyme
What is the most common substrate used for alkaline phosphatase labeled antibody?
naphthol-AS-phosphate
What chromogen is commonly used to demonstrate alkaline phosphate?
Fast red violet LB
What chromogen is INSOLUBLE in alcohol?
DAB
If 3-amino-9-ethylcarbazole (AEC) is used as the chromogen, what kind of hematoxylin should be used as the counterstain?
Mayer
Hematoxylin must not contain alcohol
How must tissue for immunofluorescence be handled?
Fresh, Frozen
DO NOT fix
Define neoplasm
New, uncontrolled and abnormal growth of tissue
Three types:
1. Benign
2. Premalignant
3. Malignant
What does a completely negative Vimentin result indicate?
Tissue has been over fixed in formalin
Besides the heat employed, what is the other most important factor of HIER?
Chemical composition
pH
How is ficin used in IHC?
A proteolytic enzyme of vegetable origin widely used for red cell antibody detection
Can be used as a trypsin substitute in immunoperoxidase staining
EIER
What is the main benefit of polymeric detection methods?
Serum and avidin-biotin blocking steps not needed
Quicker
Why must antibodies be detected with a labeled method?
Antibodies cannot be seen with a microscope unless they are labeled or flagged by some method that permits their visualization
Define the Direct-Conjugate-Labeled Antibody Method
Attaching a label to an antibody by chemical means and then directly applying this labeled conjugate to
tissue sections
The label is attached directly to the antibody with specificity for the antigen
What are the benefits of the direct conjugate labeled method?
Rapid
Ease of performance
What are the disadvantages of the direct conjugate labeled method?
Need to conjugate each of the appropriate primary antibodies separately to detect different antigens
The primary antibody be used at a relatively high concentration due to a lack of sensitivity
Define the Indirect Antibody Method
The primary antibody is unlabeled
The method uses a labeled secondary antibody with specificity against the primary antibody
a.k.a. “sandwich”
Fluorescent staining advantages
Rapid
Works well in multiplex
Non-masking
Fluorescent labels have a small molecular footprint making them easy to conjugate with an antibody
Allows analysis of proteins in fresh, frozen or fixed tissues
Can detect bacteria/parasites
Wider dynamic range
No enzyme amplification needed
Allows visualization of cell structures by super resolution microscopy
Fluorescent staining disadvantages
Fades over time
Hard to view cell morphology
Requires ability to view with fluorescent microscope
Need a specific filter to visualize each fluorophore
How can you prevent autofluorescence?
Do not use aldehydes, (formalin, glutaraldehyde) as this can result in high autofluorescence
Can be minimized by wash in sodium borhydride in PBS prior to antibody incubation
Describe autofluorescence
The natural emission of light by biological structures
Caused by flavin coenzymes and reduced pyridine nucleotides
When evaluating fluorescent-stained frozen sections, morphologic detail of tissue can be enhanced by examining slides using?
Combining fluorescent microscopy with phase contrast microscopy
A phase contrast microscope alters the phase relationships of light passing through and around an object, and as a result makes individual components (e.g. nuclei and cytoplasm) visible
What is the classic preparation of tissue for immunofluorescence?
Frozen sections of unfixed tissue
Antigenic reactivity is minimally impaired and fluorescent antibody staining is strongest
How can you combat autofluorescene?
Use probes that maximize the fluorescence signal relative to the autofluorescence
Use optical filters that maximize the fluorescence signal relative to the autofluorescence
Have a well calibrated and maintained microscope
What is fluorescence compensation?
Emission signals of the fluorphore overlap
The overlapping color must be removed or it will give a false level
The overlapping color is electronically removed using single positive controls
How can fluorescence compensation be prevented?
By using fluorophores that do not have overlapping emission spectra
What are the benefits of HRP enzymes in IHC?
Generate sharp and dense precipitates
Detects intracellular targets
Detects highly delineated locations
What are the benefits of AP enzymes in IHC?
Allows visualization of underlying tissue structure
Yield a more diffuse and translucent precipitate
What is the substrate when using HRP enzyme based detection in IHC?
Hydrogen peroxide
What is the substrate when using AP enzyme based detection in IHC?
naphthol-AS-phosphate
What two factors create staining inconsistencies when using AP enzyme based reactions?
Sensitive to heat
Sensitive to light
What chromogens are used in the HRP based detection in IHC?
DAB
AEC
What chromogens are used in the AP based detection in IHC?
Fast-red-violet LB
Fast-red TR
Fast-blue BBN
Define blocking
To block tissue endogenous peroxidase activity (needed when the tissue contains many RBCs)
To block nonspecific background staining occurring as a result of antibody attachment to highly charged collagen and connective tissue elements
How do you block for endogenous peroxide activity?
Use 3% Hydrogen Peroxide prepared in absolute methanol
How do you block for nonspecific staining?
Add nonimmune serum from the same species in which the secondary antibody is produced to the tissue before applying primary antibody
What are some examples of nonimmune proteins used for blocking?
Casein
Nondry fat milk
What wash buffers can be used with an HRP based detection system?
PBS
TBS
What wash buffer should only be used with AP based detection systems?
Use TBS only
What are the benefits of using wetting agents?
The addition of a wetting agent will help with staining
Improve rinsing steps
Slow down bacterial growth
What are some examples of wetting agents?
Tween-20
Triton-X
Brij-35
What temperature should the wash buffer be used at?
Room temperature
What are the two commonly used wash buffers?
PBS
TBS (tween)
What must be checked and maintained in the wash buffer?
pH
When must the pH of a wash buffer be checked and documented?
Each time a new lot is used
Each time a new batch is prepared
What decides the use of a certain chromogen?
The choice of chromogen is directed by the enzyme used
What are some commonly used chromogens?
DAB brown color
AEC red color
Silver black used in ISH
Fast-red fuchsia
Fuchsin fuchsia
BCIP blue
NBT blue
INT blue
What can be used to block endogenous alkaline phosphate?
Levamisole
What chromagen can be used if a deeper intense color is preferred?
Nitro BlueTetrazolium (NBT)
When using an IHC alkaline phosphatase staining method, what is the mechanism that produces the color?
An enzyme that hydrolyzes the substrate to phenolic compounds and phosphates. The phenols couple to colorless diazonium salts (chromogen) to produce insoluble, colored azo dyes.
A blue-black end product color is produced when nickel is added to which of the following substrates?
DAB
What are the two primary probe for in situ?
DNA
RNA
Explain pretreatment options in ISH
The purpose of pretreatment is to prepare the tissue section for hybridization
i.e. antigen retrieval using heat or enzymes
Explain hybridization options in ISH
The purpose of hybridization is to drive probe binding to the target - nucleic acids seek complement binding
Explain stringency wash options in ISH
The purpose of stringency wash is to remove free and nonspecifically bound probe
Destabilizes poor matches
Goal is to keep probe hybridized but remove all nonspecific binding
Encourages nucleic acids to form single strand
Explain high stringency wash in ISH
High hybridization temperatures
Low concentration of salt in the buffers
Enables the hybridization of highly homologous nucleic acid sequences
If too high, probes will not bind to the target
Explain low stringency wash in ISH
What variables are usually modified to alter the stringency in ISH?
Washing steps can be optimized by adjusting temperature, salt, and detergent concentration in the wash buffer to minimize non-specific interactions
List the steps in an ISH procedure
- Drying
- Depar
3.Pretreatment - Denaturation
- Hybridization
- Stringency wash
- Detection
- Counterstain
Identify which steps of an ISH procedure differ from an IHC procedure
IHCs do not have
1. Denaturation
2. Hybridization
3. Detection
Define enzyme
Define substrate