Small Sample SHIELD Protocol
For smaller PFA-fixed samples, the SHIELD protocol deviates from the standard protocol. In the standard protocol, samples are incubated in SHIELD-OFF Solution to allow diffusion of epoxy into the sample before it is transferred to SHIELD-ON to crosslink the epoxy. When small samples are transferred to SHIELD-ON, the epoxy can potentially diffuse out of the sample before it is cross-linked (due to their size).
In this protocol, we start with a mixture of the SHIELD-ON buffer and the epoxy to prevent diffusion when the sample is transferred to pure SHIELD-ON.
[Note] If you store the SHIELD-Epoxy in the freezer, you will need to take it out before performing SHIELD to thaw. Alternatively you can aliquot some in advance and store it at 4°C as a shorter term storage option.
Reagents Required
- SHIELD Epoxy Solution (SH-ES) – Store at -20°C
- SHIELD Buffer Solution (SH-BS) – Store at RT
- SHIELD ON Buffer (SH-ON) – Store at RT
- Temperature-controlled shaking incubator capable of 37-45°C
Protocol
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. Also, once you begin the next step, you should not stop the process until SHIELD fixation is completed, so make sure to plan ahead.
SHIELD OFF
1. Prepare fresh SHIELD-OFF Solution. Mix the solutions in the below ratios.
- SHIELD-OFF Solution: (by volume) 50% SHIELD-Epoxy, 25% SHIELD Buffer, 25% water
- We recommend making approximately 5 mL SHIELD-OFF Solution for small samples.
[Note] The following SHIELD-ON steps should ONLY be used for any sample with its smallest dimension 500 um – 1.5 mm or smaller.
SHIELD ON
3. Mix SHIELD-ON and SHIELD-Epoxy in a ratio of 7:1 to get a volume of 20 mL in a 50 mL tube
4. Incubate the sample in this solution at 37°C with shaking for 3-6 hours
- This time is dependent on tissue size: 6 hours if the smallest dimension is 1.5mm, and 3 hours if the smallest dimension is 500 µm or smaller.
5. Transfer the sample to a new conical tube with the same volume of fresh SHIELD-ON (containing NO SHIELD-Epoxy Solution)
6. Incubate at 37°C with shaking overnight.
SHIELD post-fixation is now complete. The sample can be stored in 1X PBS with 0.02% sodium azide at 4°C for several months without significant loss of fluorescence signal and structural integrity.
You may now proceed to the delipidation section of the protocol.