Perfusion fixation

From:Barry R Rittman

I think that the discussion re perfusion has missed some important
points, a few of which are mentioned here.
1. "Good fixation" of a tissue is most appropriately regarded as the
fixation that preserves the component that you are interested in and
permits the carrying out of the procedures that you wish in a consistent
manner.  As an example potassium permanganate manages to destroy almost
all the components of tissue but does retain membranes so that for this
specific purpose it can be regarded as  a good fixative.
This means that before discussing good fixation you have to define what
you wish to finally demonstrate and the methods you are going to use for
2. Perfusion versus immersion fixation. Perfusion fixation has some
distinct advantages. A good paper on the subject re electron microscopy
is by Karlsson and Schultz (1966). I am at home so do not have the
reference on hand).  The main advantage is the access of the fixative to
individual cells in a rapid manner so that cells can be killed quickly
and start to be fixed.  It generally provides superior fixation to
immersion fixation where there are many problems including diffusion
It does however have several disadvantages. All the tissues are fixed in
the same manner.  If the perfusion pressure is too high then blood
vessels can rupture and allow RBCs into the surrounding tissue. Vesssels
(such as shunts) that are normally closed may be opened.
There are usually no RBCs in capillaries making them difficult to
distinguish in some cases from lymphatic vessels. 
Perfusion is technically more difficult than immersion fixation and
requires setup of equipment. 
It is also relatively expensive, and while this may not be a problem
when using  NBF it can be for some fixing agents such as osmium
3. If you ask for a quick answer then using H and E check the details in
nuclei and the staining characteristics of the connective tissue. You
should be able to observe different nuclear details in nuclei of
different cell types, details should be sharp and the result should be
reproducible. Connective tissue should have a range of shades of pink.

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