[Histonet] fixation [of mouse kidney; long reply]

[John Kiernan]

You cannot overfix tissue structure with
formaldehyde. You can interfere with chemical
properties of the tissue. For example, storage in
formalin for many years causes loss of
stainability with eosin. 

Assuming that your PLP is the
periodate-lysine-"paraformaldehyde"
 mixture of McLean & Nakane (1974): this was
devised for preserving cell-surface carbohydrates
(glycocalyx) for EM immunohistochemistry. It
doesn't work the way the originators intended
because the lysine and formaldehyde react rapidly
in the solution, forming polymers. The polymers
may be able to form long cross-links, but they
cannot penetrate easily through cell membranes.
Interestingly, periodate alone can cross-link cell
surface glycoproteins. See Hixson et al 1981 J.
Histochem. Cytochem. 29: 561-566 for an important
study of periodate-lysine-"paraformaldehyde". The
paper is available free of charge at
http://www.jhc.org/content/vol29/issue4/
  Anyone can download the whole paper (.pdf) 
  or just the abstract of any item in the JHC 
  archives that's more than about a year old. 
Anyone intending to use PLP
(periodate-lysine-"paraformaldehyde") should read
this paper.

Returning to your mouse kidneys!
 They may be inadequately fixed, with the
glomeruli shrinking within Bowman's capsule,
perhaps an effect of the hypertonic sucrose
cryoprotectant. Intuitively, that's the simplest
explanation if your PLP is indeed
periodate-lysine-"paraformaldehyde".

BUT if you're accustomed to looking at sections of
well fixed rats' kidneys, there is a difference in
the anatomy of the glomeruli. In the mouse, the
glomerular tuft of capillaries is squat, with a
wide base that's usually in the plane of section.
Rat glomeruli are more like human ones, with the
tuft circular in section and its narrow base
(origin) visible in only a minority of sectioned
glomeruli. 

In a well fixed kidney, the great majority of
glomeruli have very little empty space between the
tuft of capillaries and Bowman's capsule. That's
supposed to be closest approximation to the
condition in life (see histology and
histotechnology textbooks etc). There are a few
lousy fixatives that inflate the glomerular tuft
and break Bowman's capsule - an obvious artifact.
See Histochemical Journal 17:1131-1146 (1985) for
some pictures of badly fixed kidneys.

John Kiernan
Anatomy, UWO
London, Canada.
--------------------------------------------
Kim O'Sullivan wrote:
> 
> Hi all,
> 
> Have recieved some mouse kidneys recently to work on which have been fixed in PLP for aproximatley 2 hours and 7% sucrose for 2 days afterwards (5 changes), but the glomeruli appear shrunken and the bowmans space enlarged (not a product of the disease we are researching). Is this caused by overfixing or underfixing?
> 
> Alternatively (as this method is quite common and we have not had this problem in the past) could this result occur throught the dehyration or rehydration steps during the staining process?
> 
> Any ideas would be helpful
> 
> Kim O'Sullivan
> 
> _______________________________________________
> Histonet mailing list
> Histonet <@t> lists.utsouthwestern.edu
> http://lists.utsouthwestern.edu/mailman/listinfo/histonet