Fixing Epithelial Organoids for Immunostaining
This protocol and volumes are optimized for whole-mount fixation of epithelial organoids (50 – 500 µm) cultured in Corning® Matrigel® (Growth Factor Reduced, Phenol Red Free, Corning Catalog #356231) domes. The epithelial organoids can be derived from different tissue types, including intestinal, mammary, prostate, lung, pancreatic, and liver derived from primary cells or pluripotent stem cells.
After fixation, perform whole-mount immunostaining of epithelial organoids:
Materials
- Anti-adherence rinsing solution
- Gentle Cell Dissociation Reagent (GCDR) OR Cold Cultrex® Organoid Harvesting Solution OR cold Corning® Cell Recovery Solution (Corning Catalog #354253)
- Axygen® 200µL Pipet Tips, Wide-Bore (Corning®, T-205-WB-C-R-S)
- 40mL fixation solution: 4% paraformaldehyde (PFA) in Phosphate-buffered saline (PBS)
- 4% PFA Solution may be aliquoted and stored at -20°C for 6 months. Avoid multiple freeze-thaws and exposure to light.
- Immunofluorescence buffer, stored at 2 – 8°C for up to 6 months
Material Quantity Final concentration PBS 499 mL BSA 500 mg 0.1 % w/v Triton™ X-100 1 mL 0.2 % v/v TWEEN® 20 0.25 mL 0.05 % v/v - Dulbecco’s phosphate-buffered saline without calcium and magnesium (D-PBS)
Tissue Sample
Recovery of Whole Organoids from Matrigel®
For organoid suspensions (i.e. grown in the absence of Matrigel® or with non-gelling Matrigel® concentrations), they do not need to be recovered, so this section can be skipped.
This recovery is optimized for organoids between 50 and 500 µm in diameter, with at least 100 organoids per panel. Some optimization may be necessary. If the organoids are less than 50 µm in diameter, start with a larger number of organoids. lf the organoids are 1 mm or larger in diameter, this protocol is not appropriate.
- Pre-rinse a 15 mL conical tube with anti-adherence rinsing solution. Tilt or vortex the tube to ensure the tube wall is coated with rinsing solution.Pre-rinsing prevents organoid adhesion to cultureware and markedly increases organoid recovery.
- Remove the culture medium from the well and add 1 mL of cold Gentle Cell Dissociation Reagent (GCDR) to the well.Cold Cultrex® Organoid Harvesting Solution or cold Corning® Cell Recovery Solution (Corning Catalog #354253) can be used in place of GCDR.
- Cut a 1 mL pipette tip (or use a wide-bore pipette tip), and pre-rinse it with anti-adherence rinsing solution. Use the same tip to (gently) triturate the dome twice, then transfer the organoids to the tube prepared in step 1.Breaking up the dome into smaller fragments allows for more efficient digestion of Matrigel®. Liberation of organoids from Matrigel® is important to prevent Matrigel® from interfering with downstream staining. Avoid excessive or harsh trituration, which may shear or damage the organoids.
- Place the tube on its side on ice. Place the ice box on a rotating or tilting platform and agitate Matrigel®-organoid suspension for 20 minutes.
- Cut and pre-rinse a 1 mL pipette tip (or use a wide-bore pipette tip) with anti-adherence rinsing solution and use the same tip to gently triturate the Matrigel® fragments.
- Place the tube back on ice and incubate for an additional 20 minutes with agitation.The incubation is complete when Matrigel® is dissolved and organoids start to float in suspension; this may require an incubation longer than 1 hour.
- Allow the organoids to settle by gravity.
- Aspirate the supernatant, which should contain most of the Matrigel®. Proceed to organoid fixation.
Protocol
There are two principal methods of chemical fixation: dehydration and cross-linking. Learn more:
The optimal fixation method should be determined experimentally for each tissue and for each antibody-antigen interaction. Generally, PFA fixation followed by antigen retrieval leads to the best results.
- Pre-rinse a 1.7 mL microcentrifuge tube with anti-adherence rinsing solution.
- Remove the supernatant from the organoid pellet and resuspend the organoids in 1 mL of 4% PFA.
- Pre-rinse a 1 mL pipette tip in anti-adherence rinsing solution and use the same tip to gently transfer the organoids from the 15 mL conical tube to the microcentrifuge tube prepared in step 1.
- Place the tube on its side and incubate in PFA with gentle rocking for 45 minutes.Although the optimal time for fixation will vary between tissues depending on their size and density (and the type of fixative), a fixation time of 45 – 60 minutes will ensure the complete fixation of organoids of most shapes and sizes (i.e. organoids up to 500 µm in diameter).
- After the incubation period, wash the organoids once in immunofluorescence buffer to eliminate residual PFA. Let the organoids settle after the wash.Performing one wash is generally sufficient. If needed, a second wash may be performed. Allow organoids to settle by gravity after all washes.
- Resuspend the organoids in 1 mL of D-PBS and proceed to antigen retrieval / immmunostaining.Organoids may be stored long term at 2 – 8°C in PBS. However, it is ideal to proceed to the next step as soon as possible to minimize loss of the antigen signal.
Notes
Safety Note
Prior to handling any chemical, consult the Safety Data Sheet (SDS) for proper handling and safety precautions.
References
- STEMCELL.com (Accessed 2024)
Performing Immunocytochemical Staining of Epithelial Organoids.
