Steps for antigen detection Flashcards
What are the possible methods in labelling antibodies? (4)
- Use of Enzymes
- Use of Fluorochrome Dyes
- Use of Colloidal Gold
- Use of Radioactive Isotopes
This is commonly used to label antibodies
Enzymatic method
Commonly used enzymes in labelling antibodies: (2)
- Horseradish Peroxidase (HRP)
- Alkaline Phosphatase
Match:
- Horseradish Peroxidase (HRP)
- Alkaline Phosphatase
a. Alternative
b. Widely used
- HRP - b. Widely used
- ALP - a. Only an alternative
This is required when using enzymes for labelling.
Chromogens
This is a color developer to produce a colored end product
Chromogens
The end product of enzymatic method can be observed using which type of microscope?
Light microscope
The use of chromogens will require the use of which substance?
Substrate
This is the one consumed by the enzyme
Substrate
Which acts as substrate in these enzymes?
- Horseradish Peroxidase (HRP)
- Alkaline Phosphatase
- HRP - Hydrogen Peroxide
- ALP - Phosphate
Technique used in Fluorochrome dyes
Immunofluoresence
Identify the following:
Horse Radish Peroxidase
Substrate:
Chromogen: 3,3’ diaminobenzidine (DAB)
Color of Chromogen:
Counterstain: (3)
Substrate: Hydrogen Peroxide
Chromogen: 3,3’ diaminobenzidine (DAB)
Color of Chromogen: Brown
Counterstain: Hematoxylin,
Methyl Green, &
Methyl Blue
Identify the following:
Horse Radish Peroxidase
Substrate:
Chromogen: 3-amino-9-ethylcarbazole (AEC)
Color of Chromogen:
Counterstain: (2)
Enumerate the following:
Horse Radish Peroxidase
Substrate: Hydrogen Peroxide
Chromogen: 3-amino-9-ethylcarbazole (AEC)
Color of Chromogen: Brick-red
Counterstain: Hematoxylin &
Methyl Blue
In HRP, hematoxylin serves as _________ counterstain, and methyl green and methyl blue as __________ counterstain.
Traditional, alternative.
Identify the following:
Alkaline Phosphatase
Substrate:
Chromogen: 5-Bromo-4-Chloro-3-Indolyl-Phosphate (BCIP)
Color of Chromogen:
Counterstain: (2)
Identify the following:
Alkaline Phosphatase
Substrate: Phosphate
Chromogen: 5-Bromo-4-Chloro-3-Indolyl-Phosphate (BCIP)
Color of Chromogen: Bluish Violet
Counterstain: Nuclear Fast Red & Brilliant Green
Identify the following:
Alkaline Phosphatase
Substrate:
Chromogen: Nitro Blue Tetrazolium (NBT)
Color of Chromogen:
Counterstain:
Identify the following:
Alkaline Phosphatase
Substrate: Phosphate
Chromogen: Nitro Blue Tetrazolium (NBT)
Color of Chromogen: Black Violet
Counterstain: Nuclear Fast Red & Brilliant Green
Identify the following:
Alkaline Phosphatase
Substrate:
Chromogen: Fast Red TR Salt
Color of Chromogen: Black Violet
Counterstain:
Identify the following:
Alkaline Phosphatase
Substrate: Phosphate
Chromogen: Fast Red TR Salt
Color of Chromogen: Rose-colored
Counterstain: None
Forms of Hematoxylin used for IHC: (2)
- Mayer’s Hematoxylin
- Harris Hematoxylin
What is the advantage of using Harris Hematoxylin?
Issue morphology are
well-defined
Form of hematoxylin with reddish-violet resulting color.
Mayer’s Hematoxylin
Form of hematoxylin with red nucleus and commonly used.
Harris Hematoxylin
Incubation time to link antibody with peroxidase:
30-60 mins
Incubation temperature to link antibody with peroxidase:
Room temperature
This label is detected using a Fluorescence Microscope
Fluorochrome Label
This label is detected using a Light Microscope
Colloidal metals
This is also used to detect antigens aside from antibodies
Lectins
Sources of lectins: (2)
Plant
Animal
Lectins can be labeled just like antibodies. These can bind only with antigens.
a. First statement is true, second statement is true.
b. First statement is true, second statement is false.
c. First statement is false, second statement is true.
d. First statement is false, second statement is false.
b. First statement is true, second statement is false.
Lectins can also bind with tissue carbohydrates
This is used in Fluorochrome label
FITC – fluorescein isothiocyanate
