TITLE: 615.1 URMC HTC Non-Inflated Fresh-Frozen Embedded Lung and Associated Tissue
AUTHORS: Gloria Pryhuber, Heidie Huyck, Lisa Rogers, Cory Poole

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Purpose      and Scope of the Procedure</div><div class = "text-block">- Rapid blocking, embedding and freezing of human lung tissue in no freezing media, 100% OCT or 5% CMC </div><div class = "text-block">- Rapid freezing of non-lung tissue in no freezing media, 100% OCT or 5% CMC</div></div>

[STEPS]
?. [Fresh Tissue Frozen in Cryomolds]
Slicing and blocking procedure should be accomplished in a grossing station or fume hood with the operator taking appropriate blood and body fluid precautions
?. [Fresh Tissue Frozen in Cryomolds]
Keep the surface of the lobes moist and cool at all times
?. [Fresh Tissue Frozen in Cryomolds]
Select lobe or partial lobes for fresh embedding and freezing
?. [Fresh Tissue Frozen in Cryomolds]
Place fresh lobe on cutting surface and section tissue into slices and further into blocks (Our standard is approximately 1cmx 1cm x  thick. See sectioning diagram for lobe in  604. URMC HTC protocol).
?. [Fresh Tissue Frozen in Cryomolds]
Work quickly to avoid warming of lung tissue.
?. [Fresh Tissue Frozen in Cryomolds]
Photograph resulting tissue sections as a map of the stored blocks. Include labels in photos to record work, location of blocks and processing method applied.
?. [Fresh Tissue Frozen in Cryomolds]
Suggest alternating slices of tissue between different embedding media. For example, a slice each to i-iii, then repeat:i.Fresh Flash Frozen Block [FFb] (not embedded in any media) - note: may not withstand long freezing period
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							ii.Fresh OCT Frozen Block 
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							iii.Fresh CMC Frozen Block
?. [Fresh Tissue Frozen in Cryomolds]
For OCT or CMC embedding: Fill the cryomolds with a thin layer of the appropriate embedding medium
?. [Fresh Tissue Frozen in Cryomolds]
Place the tissue in their respectively labeled cryomolds and position them to allow for small amount of embedding medium around the tissue
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Gently press down on the tissue with forceps in an attempt to remove as much air as possible and make sure the tissue lays flat along the bottom of the cryomold during freezing so easier to face block for sectioning
?. [Fresh Tissue Frozen in Cryomolds]
Place cryomolds containing tissue on metal pan in dry-ice ethanol bath and allow to freeze while filling the cryomold with freezing medium (OCT or CMC) to completely cover the embedded tissue.
?. [Fresh Tissue Frozen in Cryomolds]
While cryomolds are freezing, print out Freezerbondz labels for each block and place label on middle of a piece of aluminum foil.Applying the label to the foil prior to wrapping the cold blocks promotes better adhesion and reduces any warming of block while wrapping.
?. [Fresh Tissue Frozen in Cryomolds]
Once frozen, quickly wrap each corresponding block with the correctly labeled aluminum foil and return to the frozen metal pan to keep frozen until transferred to -80°C freezer
?. [Fresh Tissue Frozen in Cryomolds]
Collect wrapped cryomolds into labeled freezer bags, place in cold labeled freezer boxes and store in -80°C freezer
?. [Fresh Tissue Frozen in Cryomolds]
Prepare dry ice-ethanol (or isobutane) bath by covering bottom of flat ice bucket or styrofoam box with dry ice pellets. Pour in enough 200 proof ethanol to cover the dry ice and allow to boil. Once bubbling is stable, float a flat metal pan on the ethanol.  Add more dry ice and/or ehtanol as needed to maintain rapid freezing of molds set on the metal pan.Take care that ethanol doesn't splash over onto tissue in cryomolds.
?. [Flash Frozen Tissue for homogenates / RNA Isolation]
These tissue portions of Lung, Thymus, Spleen and Lymph Node are intended for processing for total RNA and for protein homogenates.The tissue samples are sectioned into 0.25-1.0 cm3 portions and placed in properly Freezerbondz labeled freezer vials.
?. [Flash Frozen Tissue for homogenates / RNA Isolation]
The weight of the collected tissue is recorded.
?. [Flash Frozen Tissue for homogenates / RNA Isolation]
The freezer tubes containing the individual tissues are then frozen over liquid nitrogen and placed at -800C for storage.
?. [Recording and Analysis of Results]
Complete worksheet and virtual freezer inventory
?. [Recording and Analysis of Results]
Correctly store photographs of grossing and blocking in Database