PRAC 1: ihc

Question 1

antibodies in immunohistochemsitry

diagram of antibody

how to make antibody

Answer 1

antibodies contain immunogloblin (Ig) protein fields that are also found in other proteins

antibodies contain:

• light chain
• heavy chain
• variable domains (antigen binding)
• constant region
• carbohydrate
• disulphide bonds

making an antibody:
- place protein in foreign location (i.e. animal)
- protein chopped up into peptides and presented to immune system
- isolate blood and obtain serum
best control: tissue without protein because antibody only works on tissue with protein`

Question 2

polyclonal vs monoclonal antibodies

Answer 2

polyclonal:

• many species
• antibodies bind to multiple sites on protein (better reactivity)
• multiple antigenic epitopes contained within serum

monoclonal:

• mouse and rat only
• antibody recognises single antigenic epitopes (specific tools)

Question 3

immonohistochemistry method

Answer 3

1) blocking
- reduce non-specific background
- bombard cell with lots of protein
- detergent, horse serum

2) washers
- remove anything not tightly bound
- eliminates false positive signal

3) add substrate

Question 4

antigens

Answer 4

in response to foreign object (virus) body produces many types of antibodies

antigen has many different epitopes that might be recognised by host immune system (not foreign)

b-cells recognise antigen and respond, amplify and produce antibodies

Question 5

immunolocalisations

Answer 5

principle: antigen identification in a biological sample by antibodies

direct method:

• antibody raised to recognise target antigen
• fluorochrome directly conjugated to antibody
• inconvenient because have to make specific antibody or

indirect method
- primary antibody still detects antigen

Question 6

fluorescence spectra

Answer 6

fluorescent dyes absorb best at certain wavelengths

light absorbed and emitted allows you to calculate shift = STOKES SHIFT

bigger shift = easier to distinguish signals

use filter to ignore unwanted light and increase specificity

emitting light in specific spectrum can excite a different dye = non-specific communication

Question 7

limiting factor of airy disk and microscope resolution

Answer 7

light waves have peaks and secondary waves

overlapping fluorescent dyes have low resolution

limit for microscopy is 200nm i.e. objects closer than 200nm cannot be distinguished

Question 8

fluorescent proteins (GFP)

visualising protein interactions (FRET)

Answer 8

discovery of green fluorescent protein (GFP) in jellyfish led to change in biological imaging

variations of these proteins can be attached to other proteins and cloned into cells

the GFP, attached to protein of interest, is expressed in living cells and allows us to visualise the localisation of the protein of interest in the live state

protein-protein interactions visualised by FRET

use two different fluorescent proteins so that when one is excited, energy will be transferred to the second protein via FRET provided they are sufficiently close

Question 9

types of confocal microscopy

Answer 9

point scanning: provides exceptionally high resolution in 2D and 3D

spinning disk: real time location of fluorescent reporter can be captured

two-photon: used to explore thicker samples including living samples

Question 10

sham control

Answer 10

sham control is a negative control

surgical control where the therapeutically necessary step is omitted

Question 11

roles of:

• haematoxylin
• horseradish peroxidase
• hydrogen peroxide
• eosin

Answer 11

haematoxylin stains nuclei purple.

horseradish peroxidase Converts DAB into brown precipitate.

hydrogen peroxide quenches endogenous peroxidases in tissue.

eosin stains cytoplasm pink.