Mar 20, 2026
Lightsheet Staining Protocol with Conjugated Antibodies
- Wen-hung Chou1,
- John Herndon2,
- Peter Bayguinov3,
- Feng Chen1,
- Li Ding1
- 1Washington University in Saint Louis, Department of Medicine;
- 2Washington University in Saint Louis, Department of Surgery;
- 3Washington University in Saint Louis, Department of Neuroscience
Protocol Citation: Wen-hung Chou, John Herndon, Peter Bayguinov, Feng Chen, Li Ding 2026. Lightsheet Staining Protocol with Conjugated Antibodies. protocols.io https://dx.doi.org/10.17504/protocols.io.ewov1r18ylr2/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: March 16, 2026
Last Modified: March 20, 2026
Protocol Integer ID: 313393
Keywords: Lightsheet Microscopy, FLSM, TIssue clearing, tissue samples for fluorescent lightsheet microscopy, fluorescent lightsheet microscopy, conjugated antibodies tissue processing protocol, lightsheet staining protocol, tissue sample, imaging, flsm
Abstract
Tissue processing protocol to prepare tissue samples for fluorescent lightsheet microscopy (FLSM) imaging
Materials
- Antibodies used**:
- AF594 PanCK (Abcam ab277234)
- AF488 CK5 (Abcam ab193894)
- Material Used**:
- PFA
- PBS
- Triton X-100
- Tween-20
- DMSO (Sigma D2650)
- Heparin (Sigma H3393)
- Donkey Serum
- Hoechst 33342 (40046)
Troubleshooting
Each tissue of interest is fixed in 4% Paraformaldehyde for 24 hours at room temperature.
Place the tissue in PBS and transfer it to the microscopy core for tissue clearing.
For tissue clearing, samples are infiltrated with SHIELD polyepoxy (PMID: 30556815) for 6 days with constant agitation at 4 C, were polymerized at 37 oC for 24 hours, and were then delipidated using a SmartBatch+ ETC active tissue clearing platform (LifeCanvasTechnologies, Cambridge, MA).
Samples were washed in 1x PBS for 24 hours before proceeding to antibody staining,
Transfer samples in a glass dish/vial after tissue clearing is done, since DMSO in the subsequent steps will dissolve plastic vessels.
Wash with PBS 1X with 0.2% Triton-X100, 2 times for 1 hour each at room temp.
Block tissues with PBS 1X with 0.2% Triton X-100, 10% DMSO, and 6% donkey serum for 1 day at 37°C. Use the incubator shaker at MMS8 next to the dissecting microscope (with gentle rocking ≈ 85 rpm).
Wash with PBS 1X with 0.2% Tween-20, and 10ug/mL heparin, 2 times for 1 hour each at 37°C.
Stain with conjugated antibodies in the dark except for DAPI. Antibodies are used at a 1:200 dilution with PBS with 0.2% Tween-20, 5% DMSO, 3% donkey serum, and 10 ug/mL heparin at 37°C for 4 days on a rocker or gentle shaker (≈ 85 rpm). You can stain with up to 4 conjugated-Abs with different fluorophores with excitation peaks near the laser’s wavelengths (405, 488, 561, 640), or 3 conjugated-Abs (488, 561, 640) along with nuclear staining (to be added next step).
Counter-Stain with DAPI/Hoechst on day 3. Add DAPI/Hoechst (1:10000 dilution) to the staining solution.
Wash with PBS 1X with 0.2% Tween-20 and 10 ug/mL heparin for at least 5 times for 1 hour each at room temp on a rocker or gentle shaker. Thorough washing is essential to reducing background fluorescence.
Bring tissue to microscopy core in PBS for refractive index matching and imaging.