i've rearranged this post, placing the preamble after the two questions that
I will attempt. i've noticed that netters are having posts wrongly
attributed to them (or not attributed) so i will indicate where there has
been "editing" done in the post.
> 7. Why are immunocytochemical markers believed to be characteristic of
>different cell types nearly always demonstrated in tissue culture and not in
>the intact nervous system?
Response:
1 how do you define immunocytochemical marker?
Is this antibody (mono or poly clonal) only or is in situ hybridization
included under this heading?
2 how do you define "intact" nervous system?
does this refer to whole mounted embryos (for eg) or sections of intact
nervous system dissected from animals.
3 reading between the lines, the inference is that these markers are
artefactual, only existing in T.C. conditioned cells which are only a subset
of cells form explanted tissue. i don't know if this is reasonable or not.
some gene expression studies on t.c conditioned cells versus freshly
dissected tissue do show an alteration in the number, identity of genes
expressed and a difference in the amount of expression in genes that are
turned on in both cases. this should be acknowledged but i do not think
that it invalidates the approach .
3a i suppose that another way of looking at this Q is that the markers
do not correspond to any cell in the "intact" system therfore the cells do
not exist....the expression of a marker by some cells and not others may
represent a choice of action that some cells can make in response to t.c.
"challenge ' that others cannot....the difference may be in an unnatural
system but it is still a difference, perhaps it could be useful anyway.
4 some of the reasons for starting with cultures for studying your
marker distribution is that you do work with a limited number of cell types
at a particular time, rather than having to dissect out pieces of nervous
system and treat them on the spot (ie: convenience and ease of planning of
the work). the t.c. versus in vivo argument ids not an argument at
all.....the reasonable thing to do is to use the strengths of both
approaches in a complementary manner. T.c is a good system for screening ab
and getting a "ball-park" idea of what conditions to use in vivo on whole
mounts or sections.
> 10. Why do some sections have to be 'permeabilised' to allow access of
>antibodies, since the cells are already cut open in thin sections, and one
>must assume that the permeabilisers have no effect on the immune reaction?
response:
1. yes, the cells are already cut open however the epitope that the ab
is directed against may be masked or buried by other proteins that the
epitope may interact with directly, indirectly or not at all. the
permeabilizers are used to gain access to the epitope by selectively
loosening or uncovering the epitope, presumably by selectively breaking the
interactions that mask the epitope. this is more art than science but is
still a good approach to use compared with none at all.
2. it depends if you use the permeabilisers in the immune reaction or
not...some workers use it as a pre-treatment. does it leave a permanent
effect after it is washed out...I don't know. The permeabilizers may have
an effect on the immune reaction however if you use the same reagents, it
will have a similar effect on your controls and your tests. is this a problem?
richard
At 20:25 1/09/96 GMT, you wrote:
>>NEUROBIOLOGY HAS ITS UNANSWERED QUESTIONS. HAROLD HILLMAN. SEPTEMBER 1996
>> It would be generally agreed that the identification of anomalies and
>contradictions in one's belief is an important step to advance understanding.
>Below is a series of 22 bona-fide questions, some of them drawing attention
>to contradictions. In a spirit of scientific debate, I have put forward new
>hypotheses about the histology of the nervous system, and about the mechanism
>of transmission, both of which are designed to meet the objections to the
>current views, which my questions have identified. I would request
>neurobiologists and other interested persons: (a) to answer these questions;
>(b) if they can not, to see if my theories answer them; (c) to propose their
>own solutions, if they do not agree with mine; (d) to provide some references
>to their key points; (e) to avoid rudeness and assumption of my ignorance;
>(f) to be conscious continuously of which aspects of any evidence have been
>proved, which have not, which are provable, and which are not.
>
>My references on this subject:
>(1983) Some fundamental theoretical and practical problems associated with
>neurochemical techniques in mammalian studies. Neurochem. Internat. 5, 1-13.
>(1985) The anatomical synapse by light and electron microscopy. Medical
>Hypotheses, 17, 1-32.
>(1986) Cellular Structure of the Mammalian Nervous System. MTP Press,
>Lancaster, pp 1-318.
>(1991) A re-examination of the vesicular hypothesis of transmission in
>relation to its applicability to the mammalian nervous system. Physiol. Chem.
>Phys. & Med. NMR. 23, 177-198.
>(1991) A new hypothesis for electrical transmission in the mammalian nervous
>system. Medical Hypotheses, 34, 220-224.
>(1991) The Case for New Paradigms in Cell Biology and in Neurobiology. Mellen
>Press, Lampeter, pp 1-337.
>>E-mail correspondence to: Rae West on 101722.35 at compuserve.com>>>>>>Richard Kerr.
The Murdoch Institute,
R.C.H. Flemington Rd, Parkville, 3052,
AUSTRALIA.
kerrr at cryptic.rch.unimelb.edu.au
Phone (61) 3 9345 5045.
i don't need a pithy quotation, i have enough pith, thank you.
