Embedded Files
Reagents
Reagents
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
PBS with 0.02% sodium azide (PBSN)
Wellplate
Overview
Overview
This protocol is intended for slice staining. Use this protocol if you have thin slices (anything < 500 µm) in thickness. Delipidation steps are included.
We suggest using this protocol for antibody validation. Following a standard perfusion protocol and fixation, slice the tissue with a cryostat or vibratome as normal. Check our our Megatome technology for even more slicing options! Then use this protocol to SHIELD the tissue slices.
[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.
Protocol
Protocol
[Note] If you are using cryosectioned tissue, make sure you fix the tissue with PFA before SHIELDing.
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.
[Note] The following SHIELD steps should ONLY be used for any sample with its smallest dimension 500 um and smaller
SHIELD Fixation
Mix SHIELD-ON and SHIELD-Epoxy in a ratio of 7:1, making enough to fill the well plate.
Incubate slices in this solution at 4ºC for 3 hrs in a well plate.
Move to RT and incubate at RT for ~2 hrs.
Delipidation
Move to delipidation buffer at RT or 37C for at least 3 hrs, overnight is fine as well.
Move to PBSN.
You have now completed SHIELD and delipidation, you may continue onto passive immunolabeling.
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