Re: TUNEL & PCNA methods

From:Kristel Wautier

Hello Leslie,

I will try to make a short summary.
My question on PCNA and TUNEL were as follows:

-    is it necessary to work DNase free to perform this technique?

-    the material we intend to use this technique on, needs to be
decalcified. We usually use either Decalc (on the base of HCl) or EDTA. We
wonder however, if these products could interfere with the TUNEL-detection.
Does anybody now, for example, what the effect of EDTA or HCl on DNA is?

    ANSWER1: Hello Kristel:  We perform TUNEL on undecalcified bone embedded
in paraffin all of the time and we have been doing so for several years.
First     and formost we use 5% Formic Acid for decalcification. We use
Pepsin in the technique instead of Proteinase K. We have used EDTA and it
worked also.  It is     my understanding that the acid decalcifiers open up
the double helix more so that those that are not decalcified.  The procedure
we do takes two days, we             incubated in the enzyme overnight in
the refrigerator for optimial adherence.

    ANSWER 2: 1. work as clean as possible.  The staining is very specific
for nuclei though, so I would think a DNAse contamination could be detected
and                 noticible.  DNAse treatment specifically could serve as
a positive control. Most DNAses are not real stable, as opposed to RNAse.
In any given tissue,
    it is highly unlikely that every cell would be undergoing apoptosis
(stroma, epithelium, vasculature, etc).

                        2. For decalcifying I would suggest using an EDTA
based method.  HCl can be used to denature DNA at 2M concentration, and this
might complicate interpretation.  EDTA should cause no problems on DNA
stability in fixed tissue.

                        3. Most reagents out there use fluorscein labeled
nucleotides in conjunction with terminal deoxytransferase to do the
labeling.  If your tissue has a
    high autofluorescence (can be seen with the texas red/rhodamine filter
set), there are 2 alternatives.  The first is to follow the initial labeling
with an anti-fluoroscein     antibody (available from Roche- sold as their
Tunel POD).  This antibody is coupled to an HRP.  We have been real happy
with the liquid DAB substrate sold         by Pierce.  The second way is to
use a biotinylated UTP in labeling and follow this with a strepavidin HRP.

    ANSWER 3:  have gotten consistent results with TUNEL without working
DNase free.  I have never tried it on decalcified tissue, but I'm not aware
of problems with             EDTA.  High concentrations of HCl would affect
the DNA, but I doubt the concentration in the decalcifying solution  would
affect the results of TUNEL.  Again though, I     have not tried it.

-    I have recently read that PCNA is found in two fractions in cells: a
fraction associated with the chromosomes and a fraction free in the
nucleoplasm. The latter can be "eliminated" by using an alcohol-based
fixative, prior to immunohistochemical detection of PCNA. Does anybody have
any suggestions on which fixative would be best. I have been thinking of
methacarn. Any suggestions or comments on the quality of morphology when
using alcohol-based fixatives?

ANSWER 1: We have used Histochoice fixative with good success in
paraffin-embedded sheep tissues when staining for PCNA.

ANSWER 2:  We  used the  traditional  Carnoy's without  any  pre  treatment,
but  have  latterly  moved  on  to  formalin  fixed  tissues  and  micro
waving, no   substantial  differences between the  two, and  we  did  not
find  that the presence  of  cytoplasmic staining  interfered  with
estimating proliferations etc, our  work  was  on  rodent  tissues

ANSWER 3: for the detection of PCNA in cell samples (smears, cytospins, cell
growing on slides) methanol can be used, immediate fixation in methanol,
slides can stays in methanol for several days. With this procedure we found
92% PCNA positive cells in cytospins prepared from MCF-7 cell culture

ANSWER 4: (not from histonet - personal contact): thank you very much for
your information regarding the nucleoplasmatic fraction of PCNA. Yes, we
have red         about this, but I think this will not be a larger problem
than overlabelling due to the long half-life of this protein. In each case
you have to count with over-                labelling. Overmore, there are
very big differences between the single commercial P10 anti-PCNA-antibodies
(some authors report they are not working at all on
    formalin material). On the other hand, BrDU produces under-labelling due
to its teratogenic activity . If you compare under this point of view BrDU
and PCNA,     labelling indexes should be approximately the same.

    In our work we also do not use the term "S-phase cells" but rather
cycling cells". To mark S-phases, you would need other antibodies (we re
testing some of          them, but the structures of the respective proteins
seems to be more different in different animal species, so mouse antibodies
do not work too well on our             material). For our purposes, PCNA is
sufficient, because we do not need quantitative analysis, but only to see in
situ "where it does grow".

    All the same, try out some alcoholic fixativum, you will se whether it
will be helpful. Please note that Methacarn can give false positive
    results with the peroxidase - DAB vizualization system. On the other
hand, DAB stainings are much more stable in mounted sections than
    products of APAP reaction, so you have not to take photos immediately.

To sum up: with respect to the PCNA-problem, I will compare. I will parallel
fixate in PFA, Methacarn and Carnoy. Afterwards it will become clear if
there are differences between a) alcohol or non-alcohol based fixatives, b)
methacarn or carnoy (cfr. false positives).


Kristel Wautier
Ghent University
Department of Biology
K.-L. Ledeganckstraat 35
9000 Ghent

Tel.: +32 9 264 52 31
Fax: +32 9 264 53 44

----- Original Message -----
Sent: Monday, September 23, 2002 11:46 PM
Subject: TUNEL & PCNA methods

> Hi Kristel,
>  Could you share some of these ideas with us. I have to do TUNEL and PCNA
> in mouse tissue and I have to decide about fixation and paraffin/cryo
> methods.
> Thank you,
> leslie
> Date: 20 Sep 2002 12:41:07 -0500
> From: Kristel Wautier 
> Subject: thank you note
> Hi,
> I hereby would like to thank everyone who replied to my questions on the
> assay and on the questions regarding fixation for PCNA detection.
> There were a lot of useful ideas.
> Sincerely,
> Kristel Wautier
> Ghent University
> Department of Biology
> K.-L. Ledeganckstraat 35
> 9000 Ghent
> Belgium
> Tel.: +32 9 264 52 31
> Fax: +32 9 264 53 44
> Leslie G. Ratkay DMD, PhD
> Research Scientist, Preclinical Pharmacology
> QLT Inc.
> 887 Great Northern Way
> Vancouver, BC, Canada V5T 4T5
> Phone: 604.707.7449
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