[Histonet] Help with Lung architecture

[Alejandro Ortiz Stern]

Yes, I forgot to mention that  I will be working with mice. I will be 
looking for both alveolar macrophages and dendritic cells, but my main 
concern is that although it is not a lavage per se, the instillation 
procedure might dislodge the cells on the "way in". I will look into the 
vibrating microtome to see if such an apparatus exists here, since I am 
pretty sure they will not buy one only for this particular line of research.

Thank you again for the help and the prompt response.

Gayle Callis wrote:
> 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)
>
>
>
>


-- 
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