[Histonet] Help with Lung architecture

[Gayle Callis]

I presume you are working with mice?  Since you say you have no idea about 
sucess with OCT or fixed lungs, you could try filling the lungs with OCT, 
then doing frozen sections and looking for the cells?  The same for filling 
with formalin or paraformaldehyde?  Howeer, with the latter fixation and 
wanting to do CLSM, you will have increased autofluorescence.

With lavage, you are instilling fluid then pulling the fluid with cells 
back out - basically a rinsing technic that probably mechanically dislodges 
the cells (You did not say what cells you are looking for?  alveolar 
macrophages??? ).  We stain OCT filled lungs for many kinds of cells using 
immunofluorescence staining.

You can try vibrating microtome sections on air inflated lungs, but these 
will be thicker.  One lab that specializes in respiratory diseases and 
works with mice has great success with this, and uses the autofluorescence 
levels as a counterfluorescence for cells stained using an antibody 
conjugated to red fluorophore - they obtain wonderful z stacks with this 
technic on sections that vary from 50 um and up.  They use a Leica 
vibrating microtome.

The only way I have success with air filled lungs is snap freezing the 
lungs, then cryosectioning with Instrumedics Cryojane tape transfer method 
- you can do any kind of staining after this type of cryosectioning.  The 
instrument is expensive but highly useful for problematic tissues.  You can 
go to their website and look at the instrument.

My experience with air inflated lungs and frozen sections using standard 
cryotomy is a total failure, the lungs crumble like sand and all morphology 
is lost.


At 09:21 AM 6/6/2006, you wrote:
>Hi,
>
>I am currently searching for a protocol to maintain Lung structure
>intact while avoiding intra tracheal instillation of liquids since I am
>very interested in cells that lie in the outer-most layers of the lung
>(cells that would normally become detached upon lung lavage) Or if such
>cells remain attached during paraformaldehyde instillation or OCT
>instillation that would also be very valuable information (I have no idea).
>
>If anyone has any suggestions, or knows how to fix/cut air inflated
>lungs (for example if the trachea is closed before the opening of the
>thoracic cavity or so) I would be extremely grateful for the advise.
>
>The idea is to use these sections for confocal microscopy.
>
>Thank You All in advance.
>
>--
>Alejandro Ortiz Stern M.D. Ph.D.,
>Postdoctoral Fellow
>Institut National de la Santé et de la Recherche Médicale, E0344
>Université de Nice-Sophia-Antipolis
>Institut de Pharmacologie Moléculaire et Cellulaire
>660 Route des Lucioles - 06560 Valbonne - FRANCE
>TEL: 33 (4) 93 95 77 80 - FAX: 33 (4) 93 95 77 08
>
>
>
>
>
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Gayle Callis
MT,HT,HTL(ASCP)
Research Histopathology Supervisor
Veterinary Molecular Biology
Montana State University - Bozeman
PO Box 173610
Bozeman MT 59717-3610
406 994-6367
406 994-4303 (FAX)