[Histonet] Frozen embedding of lungs

[gillian.2.brown <@t> gsk.com]

Hi Kim,
this is what we are doing for Laser Capture Microdissection.  If you are 
only doing IHC you could leave out the DEPC or even add in some protease 
inhibitors.  We find it is better to replace the air with something.  I 
have actually laid the lungs out as the pair onto their dorsal surface 
rather than cutting into pieces, it takes a little longer to freeze but we 
have no ice damage except in the muscle surrounding the oesophagus which 
has remained attached.


The thoracic cavity is opened carefully to avoid puncturing the lungs. The 
trachea is exposed by a midline neck incision and an Angiocathw catheter 
(22GA, 1IN) (Becton Dickinson Co., Franklin Lakes, NJ) is inserted in the 
trachea and tied with 4-O silk (Ethicon Inc., Somerville, NJ) to secure 
the catheter. 
By gentle pressure applied to a 1.0 cm3 syringe attached to the catheter 
the lungs are inflated with
 Tissue-Tek OCT (Sakura Finetek USA, Torrance, CA) (50% v/v) in RNase 
(DPEC [diethylpyrocarbonate])- free phosphate-buffered saline with 10% 
sucrose. The total volume instilled is, 0.6 ml. 

The lungs are removed from the thoracic cavity gently and then cut 
sagitally into several pieces ,10 mm x 10 mm, placed in the base of 
cryomolds (Sakura ), covered with additional Tissue-Tek OCT, and frozen 
immediately in liquid nitrogen-cold 2-methylbutane (usually ,10 s). The 
pieces of lung are kept flat in the bottom of the cryomold to maximize the 
area of tissue that can be sectioned.
The frozen tissue blocks are stored individually, wrapped in aluminum 
foil, at -80oC until sectioning begins. The tissue is sectioned at 7 
micron using a cryostat and a minimum of 20 sections per animal is 
prepared for LCM. The frozen sections are placed on plain glass slides (no 
adhesive) and then immediately fixed in 100% ethanol for 1 min, followed 
by rehydration through 95, 70, and 50% ethanol diluted in DPEC-deionized 
H2O (5 s each). The sections are then stained with 0.5% (w/v) Nissl 
(cresyl violet acetate) (Sigma Chemical, St Louis, MO)/0.1 M sodium 
acetate-DPEC buffer for 1 min; dehydrate in graded ethanols (5 s each); 
and clean in xylene for 5 min. The slides are allowed to air-dry 
completely and are then stored desiccated to prevent activation of 
endogenous RNase in the tissues.

Gill Brown

Target Localisation
Target Discovery
GlaxoSmithKline Medicines Research Centre,





"Kim O'Sullivan" <Kim.Osullivan <@t> med.monash.edu.au> 
Sent by: histonet-bounces <@t> lists.utsouthwestern.edu
30-Mar-2006 01:46
 
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Subject
[Histonet] Frozen embedding of lungs






Hi all,

Does any one out there have a successful method for inflating mouse lungs 
to freeze. We currently freeze them unfixed in OCT, but of course the 
morphology is shocking as the lungs have collapsed. Is there a method that 
preserves the morphololgy without collapsing or imploding the lungs.

Any advice will be appreciated

Kim O'Sullivan
Centre for Inflammatory Diseases
Department of Medicine
Monash University
Melbourne
Australia 

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