Immunohistochemistry Flashcards
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
How many different types of GABAergic interneurons are there in the hippocampus ?
12
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
What reveals newly generated cells ?
Detecting thymidine analogues
What is EDU ?
It is a thymidine analogue that is incorporated into DNA during cell division
What does PNU do ?
It increases the number of new cells
It increases cholinergic neurotransmission
What does the Fab portion of an antibody contain ?
Contains the portion which actually binds to the antigens
What does the Fc portion of an antibody tell us ?
Indicates the species in which ab is raised in
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
What are the 2 types of antibody ?
Polyclonal - harvested from serum of immunised animals
Monoclonal- single antibody produced in cultures
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
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
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
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
What is a disadvantage of indirect method ?
Secondary antibody may bind directly to tissue
What do different dyes enable ?
Different dyes have different excitation/emissions which allows for multiple labelling of different antigens in the same section
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
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
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
What does fate mapping determine ?
Determines the type of cell
What does streptomycin do ?
Enhances red fluorescence
What does DAPI stain ?
Stains the nuclei of cells
What does GFAP stain ?
It stains the astrocytes
What does NeuN stain ?
Stains mature neurons
