[Histonet] Frozen embedding of lungs
gillian.2.brown <@t> gsk.com
gillian.2.brown <@t> gsk.com
Thu Mar 30 01:56:37 CST 2006
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>
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30-Mar-2006 01:46
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[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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