>Alison Patterson wrote:
>
>I am just beginning to work with confocal, and I have some very basic
>questions.
>
>1.  Does anyone know where I can find (or does anyone have) a good
>protocol for fixing cells?  I will be using both antibodies and
>compounds like phalloidin, each labeled with fluorscein or rhodamine.
>
>2.  It seems that so many companies market these fluorescent tagged
>compounds, have you had better luck with any one supplier?
>
>Thank you in advance for any assistance.  If you feel this is too
>simple for the list (but still could help me out :-)   please feel
>free to email me directly.  Otherwise, just posting it to the list
>will bring it to my attention as well.  Thanks again, and wish me
>luck!!
>

Alison,

for the question 1), i can recommand to your attention the following books:

Glauert A.M. Fixation, dehydration and embedding of biological specimens.
North-Holland Publ. Co. and Am. Elsevier Publ. Co., 1975: In spite of this
age, this book contains very useful fixation "recipes" and general
informations about fixative effects which are still update.

Griffiths G. Fine structure immunocytochemistry. Springer-Verlag 1993:
Although intended for electron microscopy, chapters 2 and 3 of this book
should be useful for immunocytochemistry at LM level: In my opinion, it
contains the best analysis of fixative effects on cellular antigens that i
had read.

You should also find important informations in the chapter 18 of Pawley
J.B. Handbook of biological confocal microscopy (Plenum Press, 1990).

In my experience, the best fixative for fluorescence immunocytochemistry is
paraformaldehyde. We use it at 4%, freshly prepared from powder (Merck
4005-1000), in Na phosphate buffer. You should be careful about final pH
(7.2-7.4) and adapt the molarity of the buffer to your biological material
(usually between 0.15 and 0.2 M).

Good luck!


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BOUDIER Jean-Louis
INSERM U 464, Institut Federatif Jean Roche, Faculte de Medecine-secteur
nord, F-13916 Marseille cedex 20, France.
Tel (33) 491 69 88 32
Fax (33) 491 09 05 06
E-mail [log in to unmask]
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