Fixing and OCT freezing 3D hydrogels for cryosectioning

Fixing and OCT freezing 3D hydrogels for cryosectioning

Joanna Y. Lee | Chaudhuri Lab

Modified from Katrina Wisdom, originally from John Rowley

Dec. 2018

For 3D cultures of MCF10A cells embedded in rBM (i.e. Matrigel), rBM/collagen-1, collagen-1, or IPN (rBM/alginate) hydrogels in 24-well plate format.

Materials:

DMEM/F12, serum-free (cat. #11330057; Invitrogen)

16% PFA (cat. #433689; Alfa Aesar)

PBS + Ca+2 + Mg+2 (cat. #14040133; Gibco)

Sucrose (cat. #S0389; Sigma)

OCT compound (cat. #23-730-571; Fisher)

Dry ice nuggets

Small metal plate/sheet (something conductive to place on top of dry ice)

Cryomolds (cat. #62534-25; Tissue Tek)

Flat metal spatula (typically used for measuring dry chemicals)

18 gauge needle

Prepare sucrose solutions for sample dehydration:

1. 30% sucrose:

1. 30 g sucrose

2. 100 ml PBS + Ca+2 + Mg+2

2. 50/50 30% sucrose/OCT:

1. 15 g sucrose

2. 50 ml PBS + Ca+2 + Mg+2

3. 50 ml OCT (measured using 50 ml Falcon tube)

3. Autoclave. Let cool overnight.

Fixation:

4. In hood, make 4% PFA in 50 mL Falcon tube:

1. 30 mL DMEM

2. 10 mL 16% PFA

5. Warm 4% PFA in 37ºC water bath. Note: warmed to prevent rBM from dissolving.

6. In hood, use a P1000 pipetter to gently remove culture medium from gel, being careful not to pipette up gel.

7. Pipette 500 µl (1 gel volume) 4% PFA to down side of well.

8. Let incubate 30 min at RT in hood.

9. Remove 500 µl 4% PFA, being careful not to pipette up gel.

10. Wash gel with 1 ml PBS + Ca+2 + Mg+2. Note: washes using PBS + Ca+2 + Mg+2 to prevent removing Ca+2 from alginate in IPNs.

Dehydrate samples for OCT freezing:

11. Following last wash, remove PBS from gel.

12. Add 1 ml 30% sucrose to each well (keep sterile).

13. Let incubate at RT overnight.

14. Replace 30% sucrose with 1 ml 50/50 30% sucrose/OCT solution (keep sterile).

15. Let incubate at RT for 1-4 h. Note: this step aids embedded gel from separating from surrounding OCT, but longer incubations make it difficult to remove 50/50 30% sucrose/OCT from soft gels, as gel and solution begins to look homogenous.

Freeze gels in OCT:

16. Place dry ice nuggets in small Styrofoam tray (about 4 x 6 inches), shake to make as level as possible.

17. Place small sheet metal on top. Note: will make loud screeching noise.

18. Label cryomold with sample name.

19. Remove 30% sucrose/OCT from gel.

20. Fill cryomold 1/3 to 1/2  way full with OCT, avoid introducing bubbles. Place cryomold on sheet metal over dry ice laying as flat as possible.

21. Remove gel from well plate:

1. For stiff gels, use flat metal spatula to circle around edge of gel and then scoop up gel in one piece.

2. For soft gels, use a razor blade to cut off about 1/2-3/4 inch off the tip of a P1000 pipette tip. Using cut tip, pipette up soft gel.

22. As soon as OCT begins to freeze over (will go from being clear to opaque white), deposit gel into center of mold.

23. Quickly fill rest of mold with OCT, first depositing around edges of mold, then directly on top of gel. Note: for cryosectioning, it is critical that gel is completely embedded in OCT.

24. If small bubbles occur, use 18 gauge needle to scoop them up and out of the sample.

25. Keep mold on metal sheet until OCT has completely frozen solid.

26. Once frozen, place OCT embedded gel into individual zip-lock bag, remove as much air as possible when sealing, and store at -80ºC.

When ready to section, let samples equilibrate in -20ºC cryostat for 1 h, then cut 40 µm sections, 4 sections/slide. Let samples air dry, then store at -20ºC. Leftover OCT sample block should be place back into zip-lock bag with mold and returned to -80ºC.