[Histonet] Re: "chewed" Heart sections (Long)

[John Kiernan]

"Barlow, Gillian" wrote:

> This is the latest installment in our ongoing heart saga.  I previously
> wrote regarding the trashed appearance of paraffin-embedded heart sections ...
> Bottom line is, all of the slides looked fine up until the
> streptavidin peroxidase step, after which the slides again looked chewed....
> Can anyone suggest what I might be doing wrong and why we dont see this with commercially prepared slides? ...
______________________________________

Dear Gillian,

You have carried out a well controlled trial
to find the cause of your "chewed" immunostained
sections.  Here are a few more suggested lines 
of enquiry.

Did you process your own and the commercially 
prepared slides side by side in this experiment?  
If you  did, and only your sections were damaged 
by the streptavidin-HTP step, there is probably 
something wrong with way your sections are
mounted on the slides. Possible faults include:

(a) multiple tiny bubbles under the sections, 
    due to drying the mounted ribbons while 
    horizontal, before draining most of the 
    water used to float out the ribbons.
(b) poor adhesion to the glass, due to either
    a lousy adhesive or unclean glass slides.

If you didn't do yours & bought slides together,
you should do so. If the sections on the
commercial slides are also damaged by the
streptavidin-HRP solution, try exposing a few
sections to this reagent alone, omitting all
the other steps of the method, and see if they
are damaged.
  If streptavidin-HRP alone causes damage 
to both types of section, there must be
something wrong with that solution. The most
probable cause is that it its pH is far too
high. Check it with a recently (= same day)
calibrated pH meter. Check also all buffers
recently made in the lab, using bought
buffers (with sound provenances) to check the
calibration of the pH meter. 
  If your pH meter responds slowly when its
electrode is moved from one standard buffer
to another (with intervening rinses in pure
water), the electrode is probably dead. Buy
a new one. pH electrodes are expensive and 
short-lived. The meter itself is unlikely to
fail; it's electrically and electronically
a quite simple device. Don't be persuaded
to buy a new $1000s pH meter when you need 
a new $100s electrode. End of diversion.

If streptavidin-HRP alone does not cause
damage it's possible that the sections are
not adequately fixed and securely mounted, 
and damage occurs in strep-HRP because they 
have already passed through several 
potentially section-detaching steps (slightly
alkaline) and the strep-HRP was the last
straw. A comparison of your slides with the
commercially supplied ones could sort out
that possibility.

You are tackling the problem intelligently,
using the classical approach of hypothesis
and experiment. It's a method that never
completely fails, but it often raises new
questions. 
-- 
-------------------------
John A. Kiernan
Department of Anatomy and Cell Biology
The University of Western Ontario
London,   Canada   N6A 5C1
   kiernan <@t> uwo.ca
   http://publish.uwo.ca/~jkiernan/
-------------------------