DETECTION SYSTEMS Flashcards
1
Q
What is photobleaching?
A
A fluorophore loses its ability to fluorescence due to the buildup of reactive oxygen particles due to its natural activity.
2
Q
How can photobleaching be reduced?
A
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
3
Q
What are some common applications of IF in pathology?
A
- 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.
4
Q
Define ISH
A
In-situ hybridization
Technique used to identify gene amplifications, deletions and translocations as well as chromosomal copy number changes.
5
Q
What are the three different ISH techniques?
A
FISH (fluorescent)
CISH (chromogens)
SISH (silver)
Named for the system that is used to identify the probe
6
Q
Why is CISH used more commonly than FISH?
A
- 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.
7
Q
What are the two major categories of fixation?
A
Chemical
Drying
8
Q
What are the two most commonly used fluorochromes in IF?
A
FITC
Rhodamine
9
Q
What are the two most common enzymes used for antibody visualization?
A
Horseradish phosphatase (HRP)
Alkaline phosphatase (AP)
10
Q
How are DAB and AEC commonly used?
A
Visualize the sites of antibody binding by forming a colored compound
11
Q
In Avidin-biotin methods, what follows the primary antibody?
A
The biotinylated secondary antibody
12
Q
How does alkaline phosphatase function in some IHC methods?
A
As the enzyme
13
Q
Define fluorochrome
A
A dye that absorbs light then emits its own light at a longer wavelength
14
Q
How is horseradish peroxidase used in some Avidin-biotin methods?
A
As the enzyme
15
Q
What is the most common substrate used for alkaline phosphatase labeled antibody?
A
naphthol-AS-phosphate
16
Q
What chromogen is commonly used to demonstrate alkaline phosphate?
A
Fast red violet LB
17
Q
What chromogen is INSOLUBLE in alcohol?
A
DAB
18
Q
If 3-amino-9-ethylcarbazole (AEC) is used as the chromogen, what kind of hematoxylin should be used as the counterstain?
A
Mayer
Hematoxylin must not contain alcohol
19
Q
How must tissue for immunofluorescence be handled?
A
Fresh, Frozen
DO NOT fix
20
Q
Define neoplasm
A
New, uncontrolled and abnormal growth of tissue
Three types:
1. Benign
2. Premalignant
3. Malignant
21
Q
What does a completely negative Vimentin result indicate?
A
Tissue has been over fixed in formalin
22
Q
Besides the heat employed, what is the other most important factor of HIER?
A
Chemical composition
pH
23
Q
How is ficin used in IHC?
A
A proteolytic enzyme of vegetable origin widely used for red cell antibody detection
Can be used as a trypsin substitute in immunoperoxidase staining
EIER
24
Q
What is the main benefit of polymeric detection methods?
A
Serum and avidin-biotin blocking steps not needed
Quicker
25
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
26
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
27
What are the benefits of the direct conjugate labeled method?
Rapid
Ease of performance
28
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
29
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"
30
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
31
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
32
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
33
Describe autofluorescence
The natural emission of light by biological structures
Caused by flavin coenzymes and reduced pyridine nucleotides
34
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
35
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
36
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
37
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
38
How can fluorescence compensation be prevented?
By using fluorophores that do not have overlapping emission spectra
39
What are the benefits of HRP enzymes in IHC?
Generate sharp and dense precipitates
Detects intracellular targets
Detects highly delineated locations
40
What are the benefits of AP enzymes in IHC?
Allows visualization of underlying tissue structure
Yield a more diffuse and translucent precipitate
41
What is the substrate when using HRP enzyme based detection in IHC?
Hydrogen peroxide
42
What is the substrate when using AP enzyme based detection in IHC?
naphthol-AS-phosphate
43
What two factors create staining inconsistencies when using AP enzyme based reactions?
Sensitive to heat
Sensitive to light
44
What chromogens are used in the HRP based detection in IHC?
DAB
AEC
45
What chromogens are used in the AP based detection in IHC?
Fast-red-violet LB
Fast-red TR
Fast-blue BBN
46
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
47
How do you block for endogenous peroxide activity?
Use 3% Hydrogen Peroxide prepared in absolute methanol
48
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
49
What are some examples of nonimmune proteins used for blocking?
Casein
Nondry fat milk
50
What wash buffers can be used with an HRP based detection system?
PBS
TBS
51
What wash buffer should only be used with AP based detection systems?
Use TBS only
52
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
53
What are some examples of wetting agents?
Tween-20
Triton-X
Brij-35
54
What temperature should the wash buffer be used at?
Room temperature
55
What are the two commonly used wash buffers?
PBS
TBS (tween)
56
What must be checked and maintained in the wash buffer?
pH
57
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
58
What decides the use of a certain chromogen?
The choice of chromogen is directed by the enzyme used
59
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
60
What can be used to block endogenous alkaline phosphate?
Levamisole
61
What chromagen can be used if a deeper intense color is preferred?
Nitro BlueTetrazolium (NBT)
62
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.
63
A blue-black end product color is produced when nickel is added to which of the following substrates?
DAB
64
What are the two primary probe for in situ?
DNA
RNA
65
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
66
Explain hybridization options in ISH
The purpose of hybridization is to drive probe binding to the target - nucleic acids seek complement binding
67
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
68
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
69
Explain low stringency wash in ISH
70
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
71
List the steps in an ISH procedure
1. Drying
2. Depar
3.Pretreatment
4. Denaturation
5. Hybridization
6. Stringency wash
7. Detection
8. Counterstain
72
Identify which steps of an ISH procedure differ from an IHC procedure
IHCs do not have
1. Denaturation
2. Hybridization
3. Detection
73
Define enzyme
74
Define substrate
75
List the two most frequently used enzymes in IHC
76
Explain the principle of enzyme IHC methods
77
Explain the purpose of enzyme IHC methods
78
List the advantages of enzyme IHC methods
79
List the disadvantages of enzyme IHC methods
80
What is the substrate for horseradish peroxidase?
Hydrogen peroxide
81
What are the advantages of using horseradish peroxidase?
82
What are the disadvantages of using horseradish peroxidase?
83
What is the substrate for glucose oxidase?
84
What are the advantages of using glucose oxidase?
85
What are the disadvantages of using glucose oxidase?
86
What is the substrate for Beta-galactosidase?
87
What are the advantages of using Beta-galactosidase?
88
What are the disadvantages of using Beta-galactosidase?
89
What is the substrate for alkaline phosphatase?
90
What are the advantages of using alkaline phosphatase?
91
What are the disadvantages of using alkaline phosphatase?
92
Define endogenous blocking
93
Explain endogenous enzyme blocking
94
What is the microscopic result if an enzyme block is not used?
95
What is the endogenous block used for horseradish peroxidase-based detection system?
96
Where in tissues is endogenous peroxidase found?
97
What is the endogenous block used for alkaline phosphatase-based detected system?
98
Where in tissues is alkaline phosphatase found?
99
What is the blocking process for endogenous biotin that has been increased by using retrieval methods?
100
Which tissues contain high levels of endogenous biotin?
liver, kidney, pancreas, mammary gland, skeletal muscle, salivary gland and adipose tissue
101
Explain protein blocking
102
List examples of protein blocking
103
What color is the end product of 3,3'-diaminobenzidene (DAB)?
104
What are the advantages of using 3,3'-diaminobenzidene (DAB)?
105
What are the disadvantages of using 3,3'-diaminobenzidene (DAB)?
106
What are the advantages of using 3-amino-9-ethylcarbazole (AEC)?
107
What are the disadvantages of using 3-amino-9-ethylcarbazole (AEC)?
108
What color is the end product of 3-amino-9-ethylcarbazole (AEC)?
109
What color is the end product of 4-chloro-1-naphtol (CN)?
110
What are the disadvantages of using 4-chloro-1-naphtol (CN)?
111
What are the advantages of using 4-chloro-1-naphtol (CN)?
112
What color is the end product of p-phenylenediamine dihydrochloride (Hanker Yates)?
113
What are the disadvantages of p-phenylenediamine dihydrochloride (Hanker Yates)?
114
What are the advantages of p-phenylenediamine dihydrochloride (Hanker Yates)?
115
What color is the end product of Naphthol AS MX phosphate?
116
What are the advantages of Naphthol AS MX phosphate?
117
What are the disadvantages of Naphthol AS MX phosphate?
118
What color is the end product of Fast red?
119
What are the advantages of Fast red?
120
What are the disadvantages of Fast red?
121
What color is the end product of Fast blue?
122
What are the advantages of Fast blue?
123
What are the disadvantages of Fast blue?
124
What color is the end product of New fuchsin?
125
What are the advantages of New fuchsin?
126
What are the disadvantages of New fuchsin?
127
What color is the end product of 2-(p-isophenyl)-3-p-nitrophenyl-5-phenyl tetrazolium chloride (INT)?
128
What are the advantages of 2-(p-isophenyl)-3-p-nitrophenyl-5-phenyl tetrazolium chloride (INT)
129
What are the disadvantages of 2-(p-isophenyl)-3-p-nitrophenyl-5-phenyl tetrazolium chloride (INT)
130
What color is the end product of Nitro blue tetrazolium (NBT)?
131
What are the advantages of Nitro blue tetrazolium (NBT)?
132
What are the disadvantages of Nitro blue tetrazolium (NBT)?
133
What color is the end product of Tetranitro blue tetrazolium (TNBT)?
134
What are the advantages of Tetranitro blue tetrazolium (TNBT)?
135
What are the disadvantages of Tetranitro blue tetrazolium (TNBT)?
136
List four chromagens compatible with horseradish peroxidase
137
List three chromagens compatible with alkaline phosphatase
138
List three chromagens compatible with glucose oxidase
139
Identify which chromagens require special disposal
140
What methods can be used to test the reactivity of a chromagen?
141
Describe the types of specimens which may be used for immunofluorescene staining
142
What are the advantages of IF techniques?
143
What are the disadvantages of IF techniques?
144
Explain the methods used for FFPE tissue used for IF staining
145
Explain the methods used for frozen tissue sections for IF staining
146
What are the advantages of using frozen sections versus FFPE tissues for IF staining?
147
What are the disadvantages of using frozen sections versus FFPE tissues for IF staining?
148
What are the fixative options for IF staining?
149
What are the principles of fluorescent antibody techniques?
150
Explain the principles of the double layer technique
151
List the steps in the double layer technique
152
Explain the principles of the sandwich technique
153
List the steps in the sandwich technique
154
Explain the principles of the complement incubation technique
155
Lists the steps of the complement incubation technique
156
What is co-localization?
157
What is a fluorophore?
158
What is a fluorochrome?
Fluorescent dyes
159
What are the 3 fluorochomes commonly used in IF to label the anitbody?
1. Fluorescein isothiocyanate (FITC)
2. Tetramethylrhodamine isothiocyanate (TRITC)
3.
160
What color is Fluorescein isothiocyanate (FITC)?
161
What color is Tetramethylrhodamine isothiocyanate (TRITC)
162
What color is
163
What is autofluorescence?
164
List three sources of naturally occurring autofluorescence in tissue components
1. Collagen
2.
3.
165
What reduction method can be used for autofluorescence in collagen?
166
What reduction method can be used for autofluorescence in ?
167
What reduction method can be used for autofluorescence in ?
168
List the most common source of chemically induced autofluorescence
169
List the reduction methods for autofluorescence
170
Define pre-bleaching
171
Define photobleaching
172
Define CISH
173
Define FISH
174
What are the two primary main probes in ISH?
175
How do the probes differ in ISH?
176
Lists the steps in an ISH procedure
177
Which steps in the ISH procedure differ from an IHC procedure?
178
Explain pretreatment options in ISH
179
Explain denaturation options in ISH
180
Explain hybridization options in ISH
181
Explain stringency wash options in ISH
182
Define high stringency wash in ISH
183
Define low stringency wash in ISH
184
What variables are usually modified to alter the stringency in ISH?
185
What are the two aspects to blocking of background staining of tissues?
1. Nonspecific antibody binding
2. Presence of endogenous enzymes
186
What are the methods of blocking nonspecific background staining?
1. Quenching endogenous peroxidase by an H202 methanol solution
2. Blocking endogenous biotin by using the avidin-biotin blocking agent, or skim milk
3. Normal serum will act as a secondary Ab to block nonspecific binding sites
187
Examples of chromagens
Peroxidase
AEC
DAB (diaminobenzidine)
Vector S-G
Vector - VIP
Vector Nova Red
Alk Phos
New Fuchsin
Fast Red
BCIP/NBT
Vector red
Vector black
