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

[DESCRIPTION]
Purpose      and Scope of the Procedure
- Rapid blocking, embedding and freezing of human lung tissue in no freezing media, 100% OCT or 5% CMC 
- Rapid freezing of non-lung tissue in no freezing media, 100% OCT or 5% CMC

[STEPS]
SECTION: Fresh Tissue Frozen in Cryomolds
1. Slicing and blocking procedure should be accomplished in a grossing station or fume hood with the operator taking appropriate blood and body fluid precautions
SECTION: Fresh Tissue Frozen in Cryomolds
2. 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 ethanol 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.
SECTION: Fresh Tissue Frozen in Cryomolds
3. Keep the surface of the lung lobes being processed moist and cool at all times
SECTION: Fresh Tissue Frozen in Cryomolds
4. Select lobe or partial lobes for fresh embedding and freezing
SECTION: Fresh Tissue Frozen in Cryomolds
5. 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). 

SECTION: Fresh Tissue Frozen in Cryomolds
5.1. Work quickly to avoid warming of lung tissue. 
SECTION: Fresh Tissue Frozen in Cryomolds
5.2. 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.
SECTION: Fresh Tissue Frozen in Cryomolds
6. 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
ii.Fresh OCT Frozen Block 
iii.Fresh CMC Frozen Block 
SECTION: Fresh Tissue Frozen in Cryomolds
7. For OCT or CMC embedding: Fill the cryomolds with a thin layer of the appropriate embedding medium
SECTION: Fresh Tissue Frozen in Cryomolds
8. Place the tissue in their respectively labeled cryomolds and position them to allow for small amount of embedding medium around the tissue
SECTION: Fresh Tissue Frozen in Cryomolds
9. 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


SECTION: Fresh Tissue Frozen in Cryomolds
10. 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.
SECTION: Fresh Tissue Frozen in Cryomolds
11. 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.
SECTION: Fresh Tissue Frozen in Cryomolds
12. 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
SECTION: Fresh Tissue Frozen in Cryomolds
13. Collect wrapped cryomolds into labeled freezer bags, place in cold labeled freezer boxes and store in -80°C freezer
SECTION: Flash Frozen Tissue for homogenates / RNA Isolation
15. 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. 
SECTION: Flash Frozen Tissue for homogenates / RNA Isolation
15.1. The weight of the collected tissue is recorded.
SECTION: Flash Frozen Tissue for homogenates / RNA Isolation
15.2. The freezer tubes containing the individual tissues are then frozen over liquid nitrogen and placed at -800C for storage.
SECTION: Recording and Analysis of Results
16. Complete worksheet and virtual freezer inventory

SECTION: Recording and Analysis of Results
17. Correctly store photographs of grossing and blocking in Database
SECTION: Cryosectioning for Assays
14. The tissue-containing cryomolds can be sectioned as appropriate for the assay to be performed.
8-10 µm cryosections placed on glass slides for chemical stain as for H&E or low-plex immunofluorescent antibody staining.
40 µm cryosections tend to curl while being sectioned. One or more 40 µm "curls" can be placed in an RNase-free cryovial and stored at -80°C or shipped on dry ice for single nuclei isolation as in dx.doi.org/10.17504/protocols.io.ufketkw. The number of sections used is dependent on desired yield. For lung ~ 10 curls of ~ 1x1 cm tissue cryomold yields ~150-200K nuclei.
Alternating serial sections of 10 µm and groups of 10 x 40 µm "curls" are used to document the histology of the tissue from which nuclei are isolated.
SECTION: Cryosectioning for Assays
14.1. See dx.doi.org/10.17504/protocols.io.3byl4j91jlo5/v1 for further instructions re: cryosectioning fresh frozen embedded lung tissue