practical 4- immunohistochemistry

Question 1

how does direct immunofluorescence work

Answer 1

tissue section is frozen
a fluorescent agent is attached to a specific antibody (fluorescent labelled Ab)
which identifies the specific antigen (protein in tissue section)-
Ab-Ag binding
pass UV light through the section
examine using UV microscope
will be able to see where the Ag is

Question 2

what is mesangial IgA disease

Answer 2

In glomerulus

IgA is located in mesangium

Question 3

how is mesangial IgA disease diagnosed

Answer 3

a fluorescent labelled Ab to IgA is attached to the IgA showing disease is present

Question 4

name the technique that is an adaptation of direct immunofluorescence

Answer 4

indirect immunofluorescence

Question 5

how does indirect immunofluorescence work

Answer 5

same as direct immunofluorescence, but a second layer of antibody is added
more than 1 antigen can be recognised

Question 6

what is meant by multivalent

why is multivalency an advantage

Answer 6

a molecule that can attach to several other molecules

creates more/brighter fluorescence

Question 7

why is immunofluorescence important in dermatological practice

Answer 7

Ab deposition occurs in certain skin conditions

immunofluorescence allows diagnosis

Question 8

which of the following are potential drawbacks of immunofluorescence

1. frozen sections have to be used
2. difficult to see where fluorescence is located
3. difficult to see fluorescence
4. technique isnt very sensitive
5. fading of fluorescence- takes a few weeks

Answer 8

1, 2 and 5 are all drawbacks

Question 9

name the process that overcomes the drawbacks of immunofluorescence

Answer 9

immunoperoxidase

Question 10

explain roughly how immunoperoxidase works

Answer 10

similar to IF, but instead of fluorescence an enzyme is used
feed enzyme a substrate to digest
product breakdown can be reacted with a chromogen

Question 11

which is better, direct or indirect immunoperoxidase, why

Answer 11

indirect- as multivalency means that sensitivity can be increased

Question 12

explain the method for immunoperoxidase

Answer 12

Ag in tissue is embedded in paraffin wax
incubate with primary Ab
incubate with secondary Ab
add linked (multivalent molecule)
enzyme (peroxidase) attached to linker
enzyme reacts with substrate
produces breakdown product
breakdown product reacts with chromogen
chromogen produces coloured end product (usually brown) where Ag is located
analyse colour under microscope

Question 13

in the figure 3 and 4 in booklet 4, look at the tonsil micrograph

where is brown seen
what does is indicate
what colour are the nuclei
what has been used to stain the nuclei
what are the cells with brown staining

Answer 13

cell cytoplasm

site of IgG

blue

haematoxylin

plasma cells

Question 14

diagram A in booklet 4, state the localisation, distribution and intensity of the staining

Answer 14

IgG in cytoplasm

present in more than 30% of population

brown staining is strong

Question 15

how does staining help in breast tissue samples suspected of being cancerous

why is it important

in example 2 in booklet 4, state the localisation, distribution and intensity of the staining

Answer 15

they are labelled by antibodies which are attached to oestrogen receptors

decides appropriate treatment, which would be drugs that block oestrogen receptors, inhibiting growth of tumour cells

localisation- nucleus, meaning oestrogen receptors are in nucleus of cancer cells
distribution- widespread
intensity- brown staining is strong

Question 16

in booklet 4, look at example 3

explain what is happening in the picture

Answer 16

section of type of cancer found in lymph nodes/tissues

section labelled with Ab to Ag called CD3 receptor- found on T-lymphocyte surface

Question 17

in booklet 4, look at example 3

state the localisation, distribution and intensity of the staining

Answer 17

cell membrane

widespread

brown staining is strong

Question 18

which of the following cell types would be demonstrated by labelling with antibody to the substance indicated

1. muscle cells- desmin
2. thyroid C cells- thyroglobulin
3. pituitary- ACTH
4. epithelial cells- cytokeratin
5. plasma cells- fibrinogen

Answer 18

1, 3 and 4

2- need to use calcitonin instead as found in thyroid follicular cells

5- would need to use Ig instead

Question 19

what is co-localisation

is this method used now and why

Answer 19

using different enzymes, substrates and chromogens to create different colours so more than 1 antigen can be recognized

no

its easier to stain separate sections seperately

Question 20

what are the advantages of immunoperoxidase vs. immunofluorescence

Answer 20

can see where in the tissue Ag is located

completed on tissue which is embedded in paraffin wax (so slides can be labelled and preserved)