Organoid Protocol

Reagents / Equipment Required

• SHIELD Epoxy Solution (SH-ES) - Store at -20°C

• SHIELD ON Buffer (SH-ON) - Store at 4°C

• Delipidation Buffer- stored at RT

• EasyIndex – RI = 1.52 – stored at RT in sealed container

• PBSN (PBS + 0.02% Sodium azide)

• PBSTN (PBS + 1% TritonX-100 + 0.02% sodium azide)

• 32% Paraformaldehyde Solution: (EMS) 15714-1L

• A shaking incubator capable of 4°C and 37°C

Fixation

Before proceeding, please check the Expiration Date on the SHIELD-Epoxy bottle. If the solution is used after the expiration date the mechanical stability of the sample can be compromised. 

1. Fix your samples with 4% PFA using your preferred fixation method for organoids. 4% PFA isn't required, but some PFA is necessary to form the crosslinking network for SHIELD Fixation. 

2. Thaw some SHIELD Epoxy and mix it with SHIELD ON Buffer in a ratio of 1:7 (1 part Epoxy to 7 parts ON). You can perform this step with many organoids in one larger volume (~5mL) or individually with 500 µL solution in well plates or small centrifugal tubes.

3. Incubate the organoids in the mixture overnight at 4°C. 

4. Move the samples to RT and incubate for 6 more hours without changing the solution. 

At this point, the samples are SHIELD fixed and can be stored long term in PBSN at 4°C until you are ready to proceed. It is best to process samples quickly however. 

Delipidation

The exact delipidation time depends on the size of your organoids and lipid content. There are some guidelines below, but testing empirically is always best for your particular organoids. 

1. Incubate the samples in Delipidation Buffer at 37°C. Just like the fixation, this can be done with many organoids in one larger volume (~5 mL) or individually with 500 µL solution in well plates or small centrifugal tubes.

2. Use these times as a guideline based on the size of your organoid. You can always delipidate for longer than the guideline - the solution is generally gentle on tissues. 

~500 µm diameter or smaller: overnight

~1 mm diameter: 24 hours

~2-3 mm diameter: 2 days

After delipidation is complete, wash the sample in PBSTN at RT for at least 3 hours. The samples can now be stored long term in PBSN at 4°C until you are ready to proceed. It is best to process samples quickly however. 

Optional Immunolabeling

This step can be skipped if you do not need to label the samples with antibodies or dyes and are just imaging endogenous FPs. 

The exact times, temperatures, and dilutions all need to be determined empirically based on your particular sample and target of choice.

1. Prepare the primary antibody solution in PBSTN. We recommend starting with the antibody manufacturer's recommended dilution for IHC and a 3-4 day incubation at 37°C. 

2. Wash the samples for ~6hrs in PBSTN at 37°C with 4-5 refreshes of the solution. 

3. We recommend a quick (~2 hr) 4% PFA fix after primary. We have found that this reduces nonspecific aggregate signal, likely caused by primaries dissociating during secondary and forming aggregates. To do this, prepare a solution of 4% PFA with a final concentration of 1X PBS and incubate the samples at RT for 2 hours. 

4. Wash out PFA with PBSTN over several hours. 

5. Prepare the secondary antibody solution in PBSTN. We recommend using a 2:1 molar ratio of secondary to primary. Nuclear dyes like DAPI, Syto16, YoPro1, or Propidium iodide can be added to the solution to label nuclei. Please note that DAPI should only be used if the sample is ~1mm or smaller. Light penetration at that wavelength is poor even in cleared tissue. Incubate the sample at 37°C for 3-4 days. 

6. Repeat steps 2-4 to wash and PFA fix the antibodies. 

Index Matching

1. Prepare a solution of 50% EasyIndex in distilled water. Incubate the samples for 2-3 hours at 37°C. 

2. Move the samples to 100% EasyIndex and incubate for 2-3 more hours at 37°C or until transparent. 

If you can't find the samples after index matching, a small UV pen light like this one from Amazon can help find the samples.