"No distortion" fixation/cutting of lungs


Hi everyone. I'm also having difficulties with sectioning mouse lung tissue. After harvesting the left lung from the mouse, I perfuse it with a 50:50 mixture of PBS and OCT (TissueTek) through the main pulmonary artery (since we're mainly looking at blood vessels within the lung), then freeze it in OCT. When sectioning (5 micron sections), the OCT part sections perfectly, but the lung part "breaks up," and I get a lot of tissue folding at times. I thought the empty airways in the lung might have something to do with it, so I started perfusing through the airways as well. That gave some improvement in the sections, but it changed the morphology a bit. I fix in aceton after I take the slides out from -80C and air-dry them for 30 minutes. So if anyone has any suggestions on how to improve the sectioning of lungs that would be great. Thank you for your time.

Nidal E Muvarak
Associate Research Specialist
Department of Biomedical Engineering
University of Wisconsin-Madison
1550 Engineering Dr.; Rm. 2158
Madison, WI 53706-1609
Lab: (608) 265-8921; Office: (608) 265-4205; 
Home: (608) 256-7934; Cell: (608) 332-6068

----- Original Message -----
From: "Sebree Linda A." 
Date: Tuesday, April 1, 2003 4:27 pm
Subject: RE: Any tips for "no distortion" fixation and cutting of lung?

> Hi fellow UWHCer,
> Have you considered the gentle Jane freezing and tape transfer 
> method put out by Instrumedics?  I used to use it on cross 
> sections of rabbit heart. I needed adjacent sections as near 
> identical as possible to compare histochemical staining and 
> autoradiography. I believe I fixed my sections in acetone.
> If you need more info., you know where to find me.
> Linda
> Linda A. Sebree, HT(ASCP)
> University of Wisconsin Hospital & Clinics
> IHC/ISH Laboratory
> A4/204-2472
> 600 Highland Ave.
> Madison, WI 53792
> (608)265-6596
> FAX: (608)262-7174
> -----Original Message-----
> From: LaCinda Burchell [ljb@medicine.wisc.edu]
> Sent: Wednesday, March 26, 2003 10:15 AM
> Subject: Any tips for "no distortion" fixation and cutting of lung?
> Hello Histonet,
>  Investigators here wish to obtain sections of rat lung tissues with
> no (or very  minimal distortion) of rat lung representing it's 
> originalliving state.  IE. no shrinkage, compression, or 
> fractures.  We've tried
> several variations on cryoprotectants freezings and such, but have yet
> to obtain "perfect" results.  Paraffin processing is out due to
> shrinkage in processing and expansion on the waterbath.  They want 
> to be
> able to see small airways within the context of the whole rat lung 
> so EM
> seems to be out of the question.  I won't be asked to do IHC on these
> tissues so overfixation isn't an issue.  Prefusion is out of the
> question because it causes pressure changes and distortions of the
> tissue.  I'm currently considering traditional fixation techniques
> (formalin?) followed by cryosectioning.   Any suggestions or insights
> would be very appreciated.  Bless you if you can offer any help!   
> CindyB   
> LaCinda Burchell, BA, AS, HT(ASCP)
> University of Wisconsin-Madison, Medical School
> Asthma and Allergy Research IHC Lab
> 600 Highland Ave.  CSC  K4/913
> Madison, Wisconsin  53792
> Phone: 608-262-3518
> FAX:     608-263-3746

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