unanswered questions - richard kerr

Raeto West 101722.35 at CompuServe.COM
Mon Oct 7 15:35:16 EST 1996


Richard Kerr writes recently:
  "As I have finished my undergrad. training for now and am not working on 
histology/geometry of the nervous system, and harold doesn't seem to want to 
enter the debate (perhaps he just looks at the answers and uses them for his 
next hypothetical publication), I'll look on these as NOT a serious 
challenge at all, merely something to do when I like a change from TETRIS as 
my cultures do their thing. 
 
The ball is in your court, Harold/Rae/whoever you are."

OK!  Sorry about the delay, which was caused by vacations etc. I've retained
everything of your answer in what follows; however I've capitalised the
two original questions of Harold's that you answered in the hope of keeping
clarity:-


>>     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?

HH: I did not invent them or define them, but most suppliers of biochemical
reagents have pages of them. The antibodies favoured are monoclonal, but
polyclonal are also used.


>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.

HH: Both


>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.

HH: 3 & 4. The inference is *not* that the markers are 'artefactual', but they are non-specific and no-one has shown that the marking is the same in the 'intact' tissue as in tissue culture.
  Also, I do not believe that different tissues of the same animal are immunologically different, until you treat them with a host of chemicals.


>>     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.

HH: 'Masking' and 'burying' are both unprovable hypotheses. I am interested
in neurobiology in science, not art (or artefacts).


>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

HH:  What difference does it make whether it is a 'pre-treatment' or not?  *All*
treatments are 'pre-treatments', i.e. before microscopy.












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