TIPS FOR TISSUE CLEARING SUCCESS:

Submitted by wanginfo99 on Fri, 10/20/2023 - 07:39
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Applications & Tips

Sample Preparation:

  1. Fixation will vary depending on the sample type, meaning fixation time will also vary.
  2. Fixation will vary depending on the sample size. For example, small/thin tissue only requires immersion fixation, but for large tissue and organ tissue, perfusion fixation must be done before immersion.
  3. It's OK to slice the tissue after fixation (only if needed).
  4. Wash the sample(s) several times with 1X  PBS.
  5. If long-term storage is required, store the sample in 1X PBS at 4°C.

Hydrogel Infusion & Polymerization:

  1. X-CLARITY™ Polymerization Initiator can be stored for up to 6 (six) months as long as it's stored at -80°C.
  2. If the X-CLARITY™ Polymerization Initiator is stored at -80°C, it must be thawed overnight at 4°C. Best practice: Prepare hydrogel-initiator solution as needed; fresh solution provides optimal results.
  3. It's essential to use enough hydrogel-initiator solution to completely cover samples once they are submerged for incubation.
  4. If using bis-acrylamide instead of the X-CLARITY™ Hydrogel Solution, It's important to know that bis-acrylamide will form a rigid gel around tissues - this gel MUST BE REMOVED prior to clearing.
  5. If long-term storage is required, store the sample in 1X PBS at 4°C.

Tissue Clearing:

  1. Before clearing, ensure the settings of the X-CLARITY™ Tissue Clearing System match the settings designated for the sample holder being used.
  2. If using a sample holder for the first time, be sure to check samples every two hours to monitor their progression.
  3. After submerging samples, lightly tap the holder or holders to release any air bubbles that may have gotten trapped in the electrophoretic solution. If not, air bubbles could block the flow of electric current. 
  4. If storage is required, store the sample in 1X PBS at 4°C for up to one week.

Antibody labeling & imaging:

  1. During  Stage 4 of the tissue-clearing process, it's crucial to note that every step must be performed at 37°C while gently shaking.
  2. The DeepLabel™ Antibody Staining Kit offers a set of reagents optimized for clarified tissues for effective antibody penetration and site-specific binding. 
  3. If storage is required, store the sample in PBS at 4°C for no longer than two weeks.