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Experimental Skills in Neuroscience > Immunohistochemistry > Flashcards

Flashcards in Immunohistochemistry Deck (26)
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0

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

12

1

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

3 types of pyramidal cells accompanied by 21 classes of interneurons

2

What does cell fate mapping use ?

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

3

What reveals newly generated cells ?

Detecting thymidine analogues

4

What is EDU ?

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

5

What does PNU do ?

It increases the number of new cells
It increases cholinergic neurotransmission

6

What does the Fab portion of an antibody contain ?

Contains the portion which actually binds to the antigens

7

What does the Fc portion of an antibody tell us ?

Indicates the species in which ab is raised in

8

What do antibodies consist of ?

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

9

What are the 2 types of antibody ?

Polyclonal - harvested from serum of immunised animals

Monoclonal- single antibody produced in cultures

10

How can you obtain an antibody ?

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

11

What is stokes law ?

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

12

What is the indirect method ?

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

13

What are the advantages of the indirect method ?

- 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

14

What is a disadvantage of indirect method ?

Secondary antibody may bind directly to tissue

15

What do different dyes enable ?

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

16

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

We would have to punch holes in the membrane
To do this we wash the sections in buffer containing detergent

17

What controls can be used in immunohistochemistry ?

- 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

18

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

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

19

What does fate mapping determine ?

Determines the type of cell

20

What does streptomycin do ?

Enhances red fluorescence

21

What does DAPI stain ?

Stains the nuclei of cells

22

What does GFAP stain ?

It stains the astrocytes

23

What does NeuN stain ?

Stains mature neurons

24

What does PanQK stain ?

Stains oligodendrocytes

25

What does SOX2 stains ?

Stains neuronal stem cells