Immunohistochemistry

Question 0

How many types of pyramidal cells are accompanied by how many classes of hippocampal cells in the CA1 area ?

Answer 0

3 types of pyramidal cells accompanied by 21 classes of interneurons

Question 1

How many different types of GABAergic interneurons are there in the hippocampus ?

Answer 1

12

Question 2

What does cell fate mapping use ?

Answer 2

It uses thymidine analogues (BRDU &EDU) to identify newly generated stretches of DNA
- lots of DNA is made during cell division of new born cells and are loaded with thymidine analogues

Question 3

What reveals newly generated cells ?

Answer 3

Detecting thymidine analogues

Question 4

What is EDU ?

Answer 4

It is a thymidine analogue that is incorporated into DNA during cell division

Question 5

What does PNU do ?

Answer 5

It increases the number of new cells

It increases cholinergic neurotransmission

Question 6

What does the Fab portion of an antibody contain ?

Answer 6

Contains the portion which actually binds to the antigens

Question 7

What does the Fc portion of an antibody tell us ?

Answer 7

Indicates the species in which ab is raised in

Question 8

What do antibodies consist of ?

Answer 8

Consist of 4 polypeptides
- 2 heavy chains and 2 light chains joined to form a y shaped molecule
Variable region is made up of 110-130 amino acids and gives the antibody its especial th for binding antigen - it includes the ends of the light and heavy chain

Question 9

What are the 2 types of antibody ?

Answer 9

Polyclonal - harvested from serum of immunised animals

Monoclonal- single antibody produced in cultures

Question 10

How can you obtain an antibody ?

Answer 10

Immunise animal with antigen
After a few days/weeks take some blood
Centrifuge the blood to remove red blood cells
Test for presence of desired antibody

Question 11

What is stokes law ?

Answer 11

The wavelength of the fluorescent light is always greater than that of the exciting radiation but exceptions to this law have been found

Question 12

What is the indirect method ?

Answer 12

Secondary antibody raised to the gamma-globulin of the species which donated the first antibody is conjugated to the visualising molecule

Question 13

What are the advantages of the indirect method ?

Answer 13

• anti igG binds avidly
• 2 labelled secondary antibodies can bind to each primary antibody so can dilute primary antibodies and reduce contamination
• secondary antibodies are cheap and can be used to different primary antibodies as long as same species

Question 14

What is a disadvantage of indirect method ?

Answer 14

Secondary antibody may bind directly to tissue

Question 15

What do different dyes enable ?

Answer 15

Different dyes have different excitation/emissions which allows for multiple labelling of different antigens in the same section

Question 16

If a large molecule was used to label a molecule how would it pass through the membrane ?

Answer 16

We would have to punch holes in the membrane

To do this we wash the sections in buffer containing detergent

Question 17

What controls can be used in immunohistochemistry ?

Answer 17

• omit primary/secondary antibody- should get no staining
• positive control- simultaneously stain something you know should contain the antigen
• pre-adsorb antibody with antigen - if no staining occurs it tells you that the antibody does detect antigen but may also detect some others
• other antibody to different region of antigen - pattern should be the same
• western blotting- method that tells you the molecular weight of the structure that the antibody detects will also show cross reactivity with other antigens

Question 18

If PNU increases the alpha-7 subunit what will this cause ?

Answer 18

This will modulate acetylcholine and make more of it and this. Will cause neurogenesis

Question 19

What does fate mapping determine ?

Answer 19

Determines the type of cell

Question 20

What does streptomycin do ?

Answer 20

Enhances red fluorescence

Question 21

What does DAPI stain ?

Answer 21

Stains the nuclei of cells

Question 22

What does GFAP stain ?

Answer 22

It stains the astrocytes

Question 23

What does NeuN stain ?

Answer 23

Stains mature neurons

Question 24

What does PanQK stain ?

Answer 24

Stains oligodendrocytes

Question 25

What does SOX2 stains ?

Answer 25

Stains neuronal stem cells