SHIELD is a tissue preservation technique that complements and enhances the standard PFA fixation of biological tissue. It requires a series of incubation steps over the course of 4-6 days to preserve a tissue sample the size of a whole mouse brain. These steps allow epoxide molecules to diffuse into the tissue and crosslink, forming a strong skeleton that anchors proteins, enzymes, nucleic acids, and other important elements of tissue architecture. This is an ideal and necessary preparation step for active clearing or immunolabeling, as exposure to harsher chemicals or environments can strip away important targets in non-SHIELD-preserved tissue. The strong fixation of native proteins also allows for multiplex labeling, making your tissue samples even more powerful and valuable!
The SWITCH method, published in Cell in 2015, introduces a simple process for controlling a wide variety of chemical reactions during tissue processing of large animal and human samples. The method ensures uniformity of preservation and immunolabeling by synchronizing chemical reactions throughout the entire sample. There are 2 basic steps to the method: SWITCH-OFF, when chemicals and buffers are allowed to diffuse freely throughout the tissue, and chemical reactions are suppressed; and SWITCH-ON, when the buffer environment is rapidly changed to a condition allowing chemical reactivity. Tissue architecture, native biomolecules, and antigenicity are highly preserved in this method, allowing for multiple rounds (>20) of labeling.
C. DiD staining of a 1-mm-thick mouse coronal block using PBST for 1.5 days at 37°C. Only tissue surface is labeled. Scale bar, 200 μm.
D. DiD staining of a 1-mm-thick mouse coronal block using SWITCH. The sample was first incubated in DiD, 10 mM SDS containing PBS buffer for 24 hr, then moved to PBST and incubated for 0.5 day at 37°C. The whole sample is uniformly labeled. Scale bar, 200 μm.
Stochastic Electrotransport (SE), published in PNAS in 2015, is a novel method for rapid, nondestructive processing of porous samples. Processes like delipidation and immunolabeling have historically been limited by diffusion time, particularly for large samples such as an intact mouse brain. It may require weeks for antibodies to diffuse through only a few millimeters of tissue, and the labeling gradient from surface to core can be steep. SE uses a rotational electric field to disperse highly electromobile molecules (such as antibodies or surfactant micelles) throughout a porous sample without damaging electrically charged structures within the tissue. This method enables rapid clearing (2-4 days) of intact organs and rapid staining (<24 hours) with nuclear dyes, proteins, and antibodies with incredible uniformity.
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