TITLE: 624.1_URMC_HTC_Cryosectioning Non-Inflated Fresh-Frozen Embedded Lung for Nuclear Isolation or Multi-plexed In Situ Hybridization 
AUTHORS: Gloria S Pryhuber, Cory Poole

[DESCRIPTION]
This protocol describes steps to prepare non-fixed, fresh frozen tissue that has been embedded in OCT, CMC or other embedding material.
The protocol has been used in preparation for DARTFISH and MERFISH assays, as well as for representative serial section H&E stains of the tissue blocks used in these assays.

[BEFORE_START]
Label the slides or containers for the cryosections to be made as needed.

[GUIDELINES]
Care is taken to reduce RNA degradation including preventing introduction of RNases.
The optimal cutting temperature of the cryostat will depend on the tissue and the embedding material.

[STEPS]
13. Slides may be stored (briefly) at -80  but it is thought to be better to use or, if needed, ship on dry ice, within 24-48 hours of sectioning 
SECTION: Tissue Processing – Cutting Fresh Frozen Lung Sections for FISH
1. Prepare the SuperFrost Plus slides by placing them into a staining rack.
SECTION: Tissue Processing – Cutting Fresh Frozen Lung Sections for FISH
2. Rinse the slides with ultrapure water and spray them with RNaseZAP, allowing them to sit for a few minutes.
SECTION: Tissue Processing – Cutting Fresh Frozen Lung Sections for FISH
3. Rinse the slides with more ultrapure water and, while keeping them in the slide rack, dry them on the slide warmer for about 20 minutes. Keep preparing the cryostat during this time.
SECTION: Tissue Processing – Cutting Fresh Frozen Lung Sections for FISH
4. Spray the inside of the cryostat with RNaseZAP and after 3 minutes wipe down the inside with 100% ethanol making sure the chamber is dry.
SECTION: Tissue Processing – Cutting Fresh Frozen Lung Sections for FISH
5. Place a new blade into the chamber and allow to cool for 5 minutes. During this time, place the tissue block to be sectioned onto the chuck, or aside in the cryostat chamber if cutting more than one, and allow them to equilibrate to the cryostat chamber temperature
SECTION: Tissue Processing – Cutting Fresh Frozen Lung Sections for FISH
6. Place the sample (example, fresh frozen embedded in OCT, stored at -80) to be cut on the chuck and trim until a full section can be cut.
SECTION: Tissue Processing – Cutting Fresh Frozen Lung Sections for FISH
7. Cut sections at the desired thickness, generally 10 micron, and place the section onto a room temperature treated coverglass.

SECTION: Tissue Processing – Cutting Fresh Frozen Lung Sections for FISH
8. Pipette 50 µL of water from the 15 mL conical onto the correctly labeled slide for the section.
SECTION: Tissue Processing – Cutting Fresh Frozen Lung Sections for FISH
9. Place the section with the TISSUE SIDE UP onto the slide. Place into the cryostat to allow the water to freeze, adhering the section to the slide.
SECTION: Tissue Processing – Cutting Fresh Frozen Lung Sections for FISH
10. Place the slide into a slidebox on dry ice or in the chamber until finished.
SECTION: Tissue Processing – Cutting Fresh Frozen Lung Sections for FISH
11. SWITCH BLADES BETWEEN DONORS to prevent contamination of nuclei/cell material between donors.
SECTION: Tissue Processing – Cutting Fresh Frozen Lung Sections for FISH
12. Repeat until finished, keeping slides frozen throughout. Place slides in slide box equilibrated to temperature in the cryostat chamber.
14. Analysis of RNA quality of the tissue being sectioned can be achieved by isolating RNA from 2 – 20 µm tissue sections and testing for RIN and DV200.
SECTION: Tissue Processing - Cutting Frozen Tissue Curls for Single Nuclei Isolation
15. Label cryovials appropriate for the tissue section curls to be cut
SECTION: Tissue Processing - Cutting Frozen Tissue Curls for Single Nuclei Isolation
16. Spray the inside of the cryostat with RNaseZAP and after 3 minutes wipe down the inside with 100% ethanol making sure the chamber is dry.
SECTION: Tissue Processing - Cutting Frozen Tissue Curls for Single Nuclei Isolation
17. Place a new blade into the chamber and allow to cool for 5 minutes. During this time, place the tissue block to be sectioned onto the chuck, or aside in the cryostat chamber if cutting more than one, and allow them to equilibrate to the cryostat chamber temperature
SECTION: Tissue Processing - Cutting Frozen Tissue Curls for Single Nuclei Isolation
18. Place the sample (example, fresh frozen embedded in OCT, stored at -80) to be cut on the chuck and trim until a full section can be cut.
SECTION: Tissue Processing - Cutting Frozen Tissue Curls for Single Nuclei Isolation
19. Cut the tissue sections for sn isolation at the desired thickness, generally 40 micron, allowing the tissue to curl. Gently recover tissue curl into a labelled cryovial.  May place up to ~ 10 curls into a 1.5 ml cryovial. The thickness and number of sections collected per tissue block will depend on tissue type, density and desired yield.
SECTION: Tissue Processing - Cutting Frozen Tissue Curls for Single Nuclei Isolation
20. Approximately every 10 thick section, cut 1 or 2 full face, serial 10 micron sections on SuperFrost Plus slides  to stain with H&E to demonstrate morphology of the tissue being collected for sn isolation.
SECTION: Tissue Processing - Cutting Frozen Tissue Curls for Single Nuclei Isolation
21. SWITCH BLADES BETWEEN DONORS to prevent contamination of nuclei/cell material between donors.
SECTION: Tissue Processing - Cutting Frozen Tissue Curls for Single Nuclei Isolation
22. Cryovials containing cryosectioned tissue curls may be shipped on dry ice
SECTION: Tissue Processing - Cutting Frozen Tissue Curls for Single Nuclei Isolation
23. Analysis of RNA quality of the tissue being sectioned can be achieved by isolating RNA from 2 – 20 µm tissue sections or isolated nuclei and testing for RIN and DV200.