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Dear Alex:

It's hardly possible to compare immunofluorescence from different 
animals because you will have to use different primary antibodies. I 
think, comparing intensities from different parts of the same image 
should be pretty straightforward, but only from sections much thinner 
than 300 microns. Can you really see through such a thick tissue? And 
you probably cannot guarantee good antibody penetration either. I 
wouldn't use anything thicker than 20-30 microns; the thinner the 
better. Ideally, if you want to measure the mean intensity per optical 
slice, the depth of field should be less than the thickness of the 
stained layer. Without seing images, it's hard to tell how to do 
segmentation. If it's based on intensity (you say, you apply a 
threshold), you may find that the mean intensity can be very sensitive 
to the threshold level. It all depends on the image. When it is 
sensitive to the threshold, the difference between the mean and 
threshold is not, and you can use the difference - but only if you 
really want to exclude non-ER backgtround (because that's what Mean 
minus Threshold really is: staining of features over the background) 

Mike

Michael Model
Confocal Imaging Core,
Department of Biological Sciences,
Kent State University,
Kent, OH 44242
tel. 330-672-2874

----- Original Message -----
From: "Spurmanis, Aleks" <[log in to unmask]>
Date: Tuesday, January 31, 2006 9:21 am
Subject: Re: reference for quantitative immunofluorescence

> Search the CONFOCAL archive at
> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
> 
> Hi everyone, 
> 
> The below discussion came at a good time for me since I just 
> received a request to look into quantifying IF for a confocal 
> client and have limited experience on how to proceed.  The client 
> has AF594-tagged an ER-localized protein in the tubules of 300µm 
> thick rat kidney sections and would like to compare relative 
> expression in cortical vs. medullary regions within the same 
> tissue section as well as comparing the expression levels seen in 
> specimens taken from different animals.  He's expecting to confirm 
> up to 2-3 fold increases in cortical vs. medullary levels of 
> expression based on western and PCR analyses.  Given the pitfalls 
> cited below, can anyone suggest an analytical method that would 
> provide my client with reasonable (i.e., publishable) expression 
> estimates?  As a starting point, I'm considering measuring mean 
> fluorescence levels in several ROIs within the two representative 
> areas and using these to calculate an average intensity ratio for 
> each specimen.  I'm also considering thresholding the images to 
> exclude intralumenal spaces from the mean intensity calculations.  
> The image acquisition would be performed on a Zeiss LSM 510 Meta 
> (63x water lens) using a standard rhodamine detection set-up.  Any 
> comments/recommendations concerning specimen prep, acquisition set-
> up, appropriate software analysis tools (e.g., Image J plugins), 
> including relevant ref citations would be welcome.  
> 
> Thanks
> 
> Aleks Spurmanis
> Fluorescence Core Facility Manager
> Institute for Nutrisciences and Health
> 93 Mount Edward Road Suite M3-11
> Charlottetown PE
> C1A 5T1
> tel:  (902)- 566-7557
> fax: (902) - 569-4289
> e-mail:  [log in to unmask]
> 
> 
> 
> 
> 
> -----Original Message-----
> From: Confocal Microscopy List 
> [mailto:[log in to unmask]] On Behalf Of Alison North
> Sent: Thursday, January 26, 2006 1:03 PM
> To: [log in to unmask]
> Subject: Re: reference for quantitative immunofluorescence
> 
> Search the CONFOCAL archive at
> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
> 
> Hi Anda,
> 
> I totally agree with Doug about Jim Pawley's article, and I also 
> like this chapter - I know it's mainly about GFP, but the same 
> principles generally apply to IF:
> 
> Piston, D., G.H. Patterson, and S.M. Knobel. 1999. Quantitative 
> imaging of the green fluorescent protein (GFP). Methods in Cell 
> Biology 58:31-48.
> 
> Best,
> Alison
> 
> 
> Anda Cornea wrote:
> 
> >Search the CONFOCAL archive at
> >http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
> >
> >Hello all! 
> >
> >Is there a good method paper for quantitative immunofluorescence 
> that I could recommend?
> >
> >Thanks!
> >
> >Anda
> >
> >
> >
> >Anda Cornea Ph.D.
> >Head of the Imaging and Morphology Core Oregon National Primate 
> >Research Center Oregon Health & Science University
> >(503) 690-5293
> >  
> >
>