[Histonet] Question/Help on apoptosis assay

[Rich Ehrlichman]

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.
 
Rich

Judy Strauss <straussj <@t> upstate.edu> 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. 

Judy



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 compromise
the tissue? Thank you for any comments/help.

Rich




Siegel Lab
Clinical Research Building
University of Pennsylvania

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Siegel Lab
Clinical Research Building
University of Pennsylvania
		
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