Lab 3: immunohistochemical investigations Flashcards
(15 cards)
experiment procedure required to be able to view the anatomical and cellular location of antibody binding
- prepare thin sections from various regions of rat brain, mount them onto microscope slides and incubate them with one or other of the antibodies
- these “primary” antibodies will bind to their target (H1 or H2), the location of which can then be visualized by use of a “secondary” antibody carrying a chromogenic (coloured) or fluroescent marker which will recognise the original rabbit antibody
what is the difference between a primary and secondary antibody
- primary antibodies bind to the antigen of interest, with as low as possible interaction with other antigens. this latter binding would be described as “non-specific”
- secondary antibody binds to the primary antibody and carries some form of marker which allows the location to be observed
- this marker could be a colour (dye-like) staining or a fluorescent indicator. the secondary antibody also allows amplification of the signal
what does it mean if your results from the experiment showed no staining for H1 or H2
- either these substances are not present in the brain
- or that your experiment went wrong
what is positive control and what tissue could you use
positive control allows us to see if the experiment actually works
- could use liver tissue as this is where H1 and H2 are found
what does the dark staining in the images represent
“positive” staining
- indicates the location of H1 or H2 (the darker the staining the more present)
what features of H1 staining suggested it was present in neurons
- H1 staining occurs on isolated groups of cells that look like neuron (i.e. have extensive dendritic-like processes)
- higher magnification shows H1 staining in synaptic like features
what features of H2 staining suggest it is not present in neurons
- H2 staining occurs in a widely distributed population of cells, which is not a common feature for specific populations of neurons
- at higher magnification H2 stained cells are star like in shape (stellate) which matched with one type of glial cell = astrocytes
from the images, in what cell type do you think H2 could be located and how could you confirm this idea
- in order to confirm that H1 is in neurons, or H2 in astrocytes, you would need to dual label (or co-label) for an antigen that you know is selectively present in either neurons or astrocytes
- such dual labelling is often achieved using red or green fluorescent secondary antibodies
how can action potentials and other electrical properties be measured
–> can be measured in a number of experimental procedures including the use of brain slices
how are brain slices used to measure action potentials
- relatively thick sections (400um) are prepared from freshly isolated living brain tissue and transferred to a physiological buffer
- an electrode can be placed within a selected neuron and its activity recorded in response to chemical or electrical stimulation of the brain slice
- this technique relies on the cells in the brain slice remaining alive and functioning for the duration of the experiment
- to help achieve this the brain slices must be maintained in an appropriate solution called a physiological buffer
what is needed in a physiological buffer and why
- right mix of ions (Na+, K+, Cl- and ca2+)
- high Na+, low k+
- without ca2+ there will be no exocytosis, no neurotransmitter release
- total ion concentration needs to provide the right osmotic strength
- right pH
- need a buffer to maintain the pH
- right temp
- energy source (glucose) plus oxygen
- ATP won’t work because it can’t cross the cell membrane
what are neuronal cells isolated from an animal and maintained in culture used for
- provide an important resource for many neuroscience investigations
- in most cases the cells are obtained from an embryonic animal
- the tissue is carefully dissected, the cells separated from each other using a gentle enzymatic digestions of the extracellular matrix and the resulting cell suspension diluted in culture medium and transferred to a small plastic dish
- the cells will attach to the base of the dish
- culture medium contains all the ingredients the cells need to remain alive and functioning and, in some cases, grow and divide
- culture conditions can be manipulated to favour the survival of certain neuronal cell types
- such cultured cells can then be used for a variety of experimental procedures
what are the advantages and disadvantages of cultured neurons
advantages:
- provide highly controlled conditions
- regulated ion concentration
- control drug concentrations and timing
- record from individual cells
limitations:
- conditions may not be truly physiological
- lack normal contacts and interactions with other cells
- cells obtained from embryonic animals, may not develop normally
how is the concentration of ca2+ inside a cell measured
–> an increase in the conc. of intracellular ca2+ is a common neuronal response to stimulation and may occur via a number of mechanisms including the activations of neurotransmitter receptors
- measured by loading that cell with an indicator that changes its fluorescence when it binds ca2+
- this procedure is relatively simple to perform on cultured cells and the change in ca2+ concentration overtime can be recorded for individual cells
why did H1 generate a variety of ca2+ responses in the four different cells
these cultures were prepared from the rat forebrain, which contains multiple types of neurons (GABAergic, cholinergic, etc.) which are likely to respond differently to H1 and H2
