RE: [Histonet] Question/Help on apoptosis assay

From:"Connolly, Brett M"

Are you confined to doing TUNEL? If you are specifically looking for
apoptosis I would forego the TUNEL assay as my understanding is this
localizes strand breaks regardless of the mechanism (i.e. necrosis ). I
would suggest activated Caspase-3 (or maybe cleaved PARP)
immunohistochemistry on your frozen sections (20 um is OK). Or you can stain
thicker free floating sections if that is what you need for your stereology

There are a lot of good caspase-3 antibodies out there and I am sure some on
the list here can give a protocol for frozens.
Just a thought...


Brett M. Connolly, Ph.D.
Merck & Co., Inc.
MRL, Imaging Research
PO Box 4
West Point, PA 19486
PH 215-652-2501
fax. 215-652-2075

-----Original Message-----
[] On Behalf Of Rich
Sent: Thursday, June 24, 2004 10:47 AM
To: Histonet
Subject: Re: [Histonet] Question/Help on apoptosis assay

Hi Gayle,
Thanks for your info.  After showing your reply to everyone in the lab they
tell me that the blade has been sent for reconditioning recently and that
the 20 um limit had been assumed and thinner sections have never been
attempted.  So in the near future I plan on trying to cut some 10 or even 5
um sections for a few runs.  
One other question which you or someone on the list might beable to help
with is: When using frozen sections, especially with the TUNEL assay, is
there any chance we will lose some of what we are looking for?  Maybe by a
loss of structure or signal strength?  Once again I am new to all of this so
I'm going on bits and pieces of things I hear.
Thank you,

Gayle Callis  wrote:
I can't help but comment on what you wrote "I just asked about cutting 5-10
um sections with our cryostat and was told our limit for frozen sections is
20 um (possibly because our blade is not disposable)." 

I don't think your problem is that you are not using disposable blades, but
using c profile steel microtome blades that are dull! 5 um sections are
easily cut as long as c profile blades are sharp, best if the edge is newly
reconditioned. I think your limitations are directly related to a knife
that needs a good reconditioning. Obviously, that is the joy of disposable
blades, you can toss a dull blade and replace it for a wonderfully sharp

It pays to have more than one blade available, plus there are disposable
blade holders that fit in cryostat universal knife holders. I recommend you
send the steel blade(s) off to a reliable reconditioning company and enjoy
cutting thin frozen sections again. Dorn and Hart or Sturkey does this
very well. 

Hopefully, your cryostat is also in good working order, and able to cut 5
um sections. 

Good luck

Gayle Callis
Research Histopathology Supervisor
Veterinary Molecular Biology 
Montana State University - Bozeman
Bozeman MT 59717-3610

At 02:09 PM 6/23/2004 -0700, you wrote:
>Hi Judy, thank you for the quick reply. 
>I'm not sure why exactly 50 um vibratome sections were chosen. Some tests
were done to check the post assay thickness of our sections and apparently
we needed to start with 50 um. I think it has to do with the fact that we
are using a microbright stereology/morphology system and we need a certain
thickness of section. I am coming into this study already in progress so I
don't know the reasons for every choice. 
>The person who is supervising me felt that with frozen sections we would
lose some of what we are looking for. I just asked about cutting 5-10 um
sections with our cryostat and was told our limit for frozen sections is 20
um (possibly because our blade is not disposable). 
>We previously tried free floating sections but Promega kits seem to be
designed for slides only, based on the volumes of solutions it calls for.
Expanding it for use in wells would require using many kits, which is very
cost prohibitive for us during the pilot part of this experiment. 
>We are planning another run this week using a few suggested methods to
keep the sections on the slides. If this fails, I will propose your
suggestions. Thank you again.
>Judy Strauss wrote:
>Hello Rich,
>Greetings from Syracuse, NY. My daughter is currently at Wharton in the
>MBA program. 
>Is there any reason why you are using 50 um vibratome sections? I would
>suggest cutting your formalin fixed brains at 5-10 microns on a cryostat. 
>The thinner sections will adhere to the slides and the TUNEL reagents will
>penetrate the tissue. Thick vibratome sections are going to present other
>than fall off problems, ie. reagent penetration, permeabilizing cell
>membranes, etc. If you must use thick vibratone sections you might try the
>TUNEL process free floating and mount the tissue sections on slides at the
>very end. 
>Judith Strauss
>SUNY Upstate Medical University
>Department of Orthopedic Surgery
>IHP room 3118
>505 Irving Avenue
>Syracuse, NY 13120
>phone: (315) 464-9960
>fax: (315) 464-6638
>>>> Rich Ehrlichman 06/23/04 01:09PM >>>
>Hello all, I am new to this list and also new to histology in general. 
>This list seems like a very useful tool for anyone doing histology so I
>thought I'd give it a shot. I'm a student working in a lab at U Penn. We
>are currently working on staining for apoptosis in mouse brains using the
>Promega Tunel kit. They are fixed for 24 hours in 10% buffered formalin and
>we have been trying to use fresh vibratomed sections cut at 50 um. We are
>having a problem with the sections coming off of the slides during the
>assays. They wouldn't stick to slides we had gelled ourselves so on a
>suggestion from someone with more experience we purchased ultra stick
>slides. These didn't work any better. We basically floated the sections,
>let them sit in a humidified chamber for about 5 minutes and then started
>the assay. It has been suggested that we should let them sit over night in
>the humidified chamber or possibly use heat to get them to stick. I'm
>having trouble finding out whether that would
>be a problem for the tissue since these are not in paraffin. So basically
>my question is, does anyone have any tips or experiences with getting the
>sections to stay put or just general methods we can try that wont
>the tissue? Thank you for any comments/help.
>Siegel Lab
>Clinical Research Building
>University of Pennsylvania
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>University of Pennsylvania
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