Cryosectioning#

Materials & Reagents#

  • Sucrose (powder)

  • OCT compunds

Solution preparation#

  • Prepare 30% sucrose solution:

Reagent

Volume

DI water

350 g

Sucrose

150 g

Total

500 ml

  • Prepare 20% sucrose solution:

Reagent Volume
DI water 400 g
Sucrose 100 g
Total 500 ml

  • Filter the both solution (vacuum bottles with 0.2um filter)

Protocol#

Organoid preparation (sucrose gradient)#

  1. Incubate fixed organoids in sucrose 20% overnight

  2. Incubate in sucrose 30% for > 1h

  3. Take either a 6 or 12 well plate This depends on the number of different samples you are going to embed

  4. Fill one well (or the first of each row) with 30% sucrose solution

    Note: Do not fill all the way, just enough to submerge the largest organoid

  5. Fill the next two wells (or the next two of each row) with OCT

    Note: Do not fill all the way, just enough to submerge the largest organoid

  6. Label a cryomold with the following

  7. Fill the cryomold with OCT

    Note: Do not overfill otherwise OCT will drip over the edges

  8. Make sure organoids in the sample tube are all free (none sticking to the bottom)

  9. Quickly dump organoids into the well filled with 30% sucrose

    Note: If an organoid gets stuck in the tube, take some sucrose from the well with a dropper, and place in tube to try to free the stuck organoid. Try to dump out the remaining organoid with the sucrose solution

  10. After washing organoids in 30% sucrose, transfer each individually using microdissection forceps to the 1stwell filled with OCT

    Note: Do not pinch organoid with forceps, rather place forceps underneath and lift.

  11. Gently wrap organoids in OCT, ensuring all surfaces are covered

  12. Move organoids to second OCT well and repeat

  13. Use forceps to place each organoid into the sample plate

  14. Place organoids on the surface, avoiding touching forceps to OCT

    Note: Do not place organoids too close to the edge

    Tip: place smaller organoids first as they sink slower, while bigger organoids sink faster and could reach the bottom of the plate too soon. Don’t put more than 10/12 organoids in each mold.

  15. Use forceps to push down appropriate organoids so that all organoids are on same plane

  16. Once all organoids are submerged (but not at bottom), place sample plate in -80C freezer

Cryosectioning#

Cryostat: Leica CM2050 S Manual: https://www.americaninstrument.com/pdf/1779E-CRYOSTAT.pdf

Cryostat settings:

    Thickness 10-25 um 
    Temperature approx. -20 C

Preparing cryostat:

  1. Place sample on flat surface inside cryostat (such as quick freeze surface (8)) while setting up

  2. Place blade and Flip knife guard (if not done so already)

  3. Rotate lever located directly right of stage to loosen blade holder

  4. Place blade onto knife holder and slide in

  5. Check that blade is evenly placed

  6. ush lever to tighten knife holder

  7. Add OCT and place sample Add approx. ½ tablespoon of OCT onto specimen disk and quickly place sample, maintaining correct orientation Add weight and let sit for >10 minutes

  8. Prepare slides Lay out 20 slides on slide book Label 1st slide with:

  9. Use single down arrow or wheel to move specimen close to blade.

    Note: Begin by slicing/trimming OCT from top of sample (make sure anti-roll glass plate is flipped up)

  10. Adjust sample angle appropriately to achieve even, whole slices

  11. Brush away debris as needed

  12. Once organoids become visible…

  13. Replace glass anti-roll plate

  14. Slowly slice to minimize sample rolling

  15. Remove anti-roll glass plate slowly

  16. Take test slide, and flip face down vertically, with label closest to specimen head

  17. Delicately hold slide close to sliced specimen to allow specimen to adhere to the slide

  18. Examine slide for organoids

  • i. If none appear, keep trimming and perform another test Once slices are whole and even, and organoids are apparent, add sample slices to slides Add one slice to slides 1-20, followed by a second and then third. Trim 2-4 times between taking samples

  • i. Trim more between samples if majority of organoids are in different planes in OCT

  • ii. Trim less between samples if organoids are at same level in OCT

Cleaning

  1. Remove specimen disk and remaining specimen and let thaw on paper towel

  2. Remove specimen from disk and clean disk with 70% EtOH

  3. Dry and place back inside cryostat

  4. Brush as much debris as possible into removable debris tray

  5. Quickly remove tray and dump contents into biohazard bin (OCT will melt and stick instantly)

  6. Wash debris remnants with 70% EtOH and let tray dry

  7. Replace tray

  8. Spray paper towel with 70% EtOH and clean remaining debris inside cryostat DO NOT TURN CRYOSTAT OFF

Immunostaining Protocol#

  1. Prepare TBS-T + 3% BSA solution

    • 6g of BSA in 200mL of TBS-T 1x (BSA powder, Millipore Sigma A7906-100G, stored in iPSC 4C fridge)

    • BSA takes a little while to dissolve, so do this step early

  2. Incubate slides in 1x Tris Buffer Solution – Triton (TBS-T) for 10 minutes

  3. Incubate slides in the TBS-T + 3% BSA solution for 30min

  4. Prepare primary antibody solution

    • 250uL/slide * (n slides) = _X_mL TBS-T + 3%BSA

    • Add appropriate volume of antibody for desired dilution

    *keep primary antibody in ice bucket and return to fridge immediately

  5. Add primary antibody solution to slides

  6. Remove extra solution by tapping slide side on paper towel

  7. Wipe edges of slide with kimwipe

  8. Outline slide lightly with hydrophobic marker (PAP pen)

  9. Place slides in large dish with wet kimwipes (2-4/dish) to maintain humidity

  10. Transfer 250uL of primary antibody solution to conical microtubes (one for each slide)

  11. Transfer primary antibody solution from conical microtube to sample slide using dropper (do not use 1000uL pipets as flow could detach the organoid slices)

  12. arefully tilt slides to spread solution evenly over slide

  13. Place dish lid and let sit overnight in 4C fridge

  14. Wash slides with 1xTBS-T for 10min

  15. Prior, remove extra primary antibody solution by tapping slide edges on paper towel

  16. Prepare secondary antibody solution

  17. Centrifuge secondary antibody

  18. Add appropriate volume of secondary antibody to X mL of TBS-T + 3% BSA for a 1:500 dilution

  19. Add secondary antibody solution to slides

  20. Remove extra TBS-T by tapping slide side on paper towel

  21. Place slides back in large dishes with kim wipes

    Note: *If necessary, re-outline the slide with hydrophobic marker (usually not necessary)

  22. Transfer 250uL of secondary antibody solution to conical microtubes (one for each slide)

  23. Transfer secondary antibody solution from conical microtube to sample slide using dropper (do not use 1000uL pipets as flow could detach the organoid slices)

  24. Carefully tilt slides to spread solution evenly over slide

  25. Cover dishes with aluminum foil to protect from light, and let sit for 1hr at RT

  26. Wash with TBS-T for 10min

  27. Mount slides

  28. Let slides partially dry on paper towel

    Note: *If necessary, re-outline the slide with hydrophobic marker

  29. Add mounting media (~3 drops per slide) with DAPI and spread by tilting slide

  30. Add coverslips and let sit for 10-15min or until mounting media is dry

    Make sure to cover to protect from light

  31. Seal with nail polish

  32. Store in -20C freezer, image the following day.

    Quality of the staining will degrade quickly so image all within a few days/ one week and then discard the slides in sharp biohazard container.