Tissue-Tek* OCT

Medium such as Tissue-Tek OCT media (Baxter Diagnostics, McGaw Park, IL) or gum tragacanth (Fisher Scientific, Pittsburgh, PA) 7% in H2O. 

For tissue collection, take samples according to the method of analysis snap-freeze portions of kidney in liquid nitrogen for RNA extraction, roll tissue in Tissue Tek OCT (Cryoform, IEC, Needham, MA) and snap freeze in isopentane cooled over dry ice fix tissue in 4% paraformaldehyde for in situ hybridization or in 10% buffered formalin for routine histology. 

Cryofixation is done by the immersion of tissues in ice-cold isopentane for 3 min followed by freezing at -80°C. Fixed, frozen tumors are mounted in Tissue-Tek OCT 4583 compound (Sakura Finetek, Torrance, CA) and sectioned on a Microtome Plus (TBS). 

Embed the gels m Tissue-Tek OCT compound using a ciyomold. 

Embed tissue in Tissue-Tek OCT compound and freeze with CO2... 

Align the slices in a plane, remove excess fluid, and freeze them in Tissue-Tek OCT compound using CO2 (see Note 24). 

Optimum cutting temperature (OCT) moimting medium, e.g., Sakura Tissue-Tek OCT Compound (Gentairr, San Jose, CA). 

OCT Tissue Tek from Miles Laboratories wax pen (Marktex, Tech Pen, Scientific Products, McGraw Park, 111.), or PapPen (SCI, Science Services, Munich, Germany). 

Mount specimens on a cutting chuck with OCT mounting medium (Tissue Tek II, Miles) in a cryostat chamber and cool until the OCT solidifies. 

OCT (optimal cutting temperature) compound (Tissue-Tek, ProSciTech, Thuiingowa). 

Fill Tissue-Tek cryomolds with OCT medium and place a piece of tissue in each labeled cryomold. Minimize the number of bubbles and avoid bubbles in contact with the tissue. 

Sampling The infected intra-cerebrally mice for Obihiro strain (3) and hamsters for Sc237 (4) at each infected stages were sent from PDRC/NAHI (Ibaraki, Japan) after all the safety precautions for the study were fulfilled. Brain tissues (hippocampus domain) were sampled under P2 level conditions and were embedded adjacently with normal brain tissues (hippocampus domain) into an embedding medium for frozen tissue specimens (OCT Optimal Cutting Temperature Tissue-Tek, Sakura Fine-technical Co., Ltd., Tokyo, Japan), respectively. 

Tissue IS removed and placed on a brass chuck with surrounding OCT compound (Lab-Tek Products, Naperville, IL) or brain paste and then quickly frozen in liquid nitrogen or on dry ice. The tissue may first be perfused in vivo by a light fixative, such as 0.1% formaldehyde and/or 5-15% sucrose to improve histological features if the receptor is not affected. After freezing, the tissue is stored at — 70°C until used. 

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