IMMUNOHISTOCHEMISTRY

Question 1

Identification of specific tissue or cellular antigens, found on tissues

Answer 1

Immunohistochemistry

Question 2

Used for detection of organisms in cytologic preparations (fluids, sputum, FNA)

Answer 2

Immunohistochemistry Application

Question 3

Most commonly used antibody class

Answer 3

IgG

Question 4

Produced by different cells, reacts with various epitopes, common source rabbit

Answer 4

Polyclonal Antibodies

Question 5

Other animal sources for polyclonal antibodies

Answer 5

Goat, pig, sheep, horse, guinea pig

Question 6

Source of monoclonal antibodies

Answer 6

Mice

Question 7

Storage of aliquots in immunohistochemistry

Answer 7

-80°C

Question 8

Enzyme labels in Immunohistochemistry

Answer 8

HRP (Horse Radish Peroxidase) and AP/ALP (Alkaline Phosphatase)

Question 9

Common chromogens

Answer 9

DAB (Diaminobenzidine), AEC (brick red), Nitroblue tetrazolium, Fast Red TR salt

Question 10

Preferred chromogen for permanent stains

Answer 10

DAB

Question 11

Chromogen for skin sections

Answer 11

AP Red (to prevent melanin masking)

Question 12

Double staining method

Answer 12

DAB and AP in the same section

Question 13

Incubation time for antibody-peroxidase linkage

Answer 13

30-60 minutes at room temp

Question 14

End product visualization

Answer 14

Light microscopy

Question 15

Fluorochrome used in IHC

Answer 15

FITC (Fluorescein Isothiocyanate)

Question 16

Requirement for fluorochrome detection

Answer 16

Fluorescent microscope

Question 17

Lectins definition

Answer 17

Plant/animal proteins binding to tissue CHO, can be labeled like antibodies

Question 18

Fixation for frozen sections

Answer 18

Absolute methanol or acetone

Question 19

Fixation for paraffin embedded tissue

Answer 19

10% Neutral Buffered Formalin

Question 20

Other fixatives used

Answer 20

4% paraformaldehyde, Bouin’s, Zamboni’s

Question 21

Unmasking requirement

Answer 21

For paraffin embedded tissues before IHC

Question 22

Common counterstains

Answer 22

Hematoxylin, Methyl green, Nuclear fast red

Question 23

Adhesive for tissue sections

Answer 23

APES diluted in acetone

Question 24

Inactivators for blocking enzymes

Answer 24

3% Hydrogen Peroxide for HRP, Levamisole for ALP

Question 25

Blocking agents purpose

Answer 25

Animal serum to block non-specific binding

Question 26

Blocking time

Answer 26

10-30 minutes at room temperature

Question 27

Mounting medium requirement

Answer 27

Neutral pH, 50% glycerol for short-term storage

Question 28

DAB; Counterstain: Hematoxylin, Methyl Green, Methyl Blue; Mounting Medium: Neutral

Answer 28

HRP with DAB

Question 29

AEC; Counterstain: Hematoxylin, Methyl Blue; Mounting Medium: Water Soluble

Answer 29

HRP with AEC

Question 30

BCIP/NBT; Counterstain: Nuclear Fast Red, Brilliant Green; Mounting Medium: Neutral

Answer 30

AP with BCIP/NBT

Question 31

Fast Red; Counterstain: Hematoxylin, Methyl Green, Brilliant Green; Mounting Medium: Water Soluble

Answer 31

AP with Fast Red

Question 32

Tissue preparation

Answer 32

Deparaffinization > Inactivation using 3% hydrogen peroxide > Antigen retrieval > Blocking > Application of antibody > Staining > Mounting

Question 33

A section known and proven to have the antigen in question

Answer 33

Positive control

Question 34

Omit the primary antibody from the staining schedule. Replace the specific primary antibody with an Ig directed against an unrelated antigen

Answer 34

Negative control

Question 35

Contains the target antigen but not in the tissue elements under investigation

Answer 35

Internal tissue control

Question 36

Extended Polymer One Step Staining; direct labeling system with primary Ab conjugated to labeled long-chain dextran polymer

Answer 36

EPOS

Question 37

More sensitive than direct; increases signal amplification using labeled secondary Ab

Answer 37

Two-step indirect technique

Question 38

Secondary Ab conjugated to enzyme-labeled polymer chain

Answer 38

Polymer Chain Two-step indirect technique