We want to look at the 3D architecture of pulmonary vasculature - the thicker the sections, the more information, easier to follow branching patterns, etc. - especially with a confocal microscope. I would like to cut sections which are several hundred microns thick. The only way I can think of doing this is with a vibratome, using unembedded tissue. But I do worry about the effect of prolonged fixation on antigenic sites.
>We routinely store in 70% ethanol(ETOH). High enough concentration of ETOH
>for most things not to grow but not so high as to cause the tissue to
>become too brittle. Of course we dont do this indefintely. What are the
>concerns over embedding? A lot of the problems that arise during
>immunostaining are caused by the fixation not necessarily the embedding
>procedure and in that case I would probably freeze half and fix the other
>I have whole lung tissue, from rat, obtained by perfusion with 4%
>paraformaldehyde, removed, and then further fixed in PFA [that's
>paraformaldehyde ;-) ] for 3 days. The plan is to collect thick vibratome
>sections, so the tissue will never be embedded. I would like to keep part
>of the lung for sectioning in the future and to carry out
>immunocytochemistry. What solution should I keep them immersed in? Right
>now they are in PBS, at 4deg C but I feel that some preservative should be
>added to prevent decomposition. However, keeping them in fixative may
>reduce their antigenicity.
>I would appreciate hearing any suggestions from this very knowledgable
St. Michael's Hospital, 8Q
30 Bond St.
Toronto, ON M5B 1W8
ph: 416-864-6060 x6337
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