Apr 30, 2026
Tissue clearing protocol
- Eunkyung Park1
- 1Beth Israel Deaconess Medical Center
Protocol Citation: Eunkyung Park 2026. Tissue clearing protocol. protocols.io https://dx.doi.org/10.17504/protocols.io.4r3l2dkbqg1y/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: April 30, 2026
Last Modified: April 30, 2026
Protocol Integer ID: 315991
Keywords: tissue clearing protocol, tissue clearing protocol this protocol, accelerated tissue clearing method, tissue clearing method, clearing of intact human organ, analysis of smaller tissue specimen, smaller tissue specimen, scalable tissue labeling, clearing, resolution 3d imaging, intact human organ, miltenyi ultramicroscope blaze, sized sample, tiff generation, rapid processing, routine laboratory application
Funders Acknowledgements:
U54
Grant ID: U54HL165440
Abstract
This protocol outlines a simplified and accelerated tissue clearing method specifically modified for small-sized samples, inspired by established Nature Protocols. The procedure has been optimized to significantly reduce overall incubation times and streamline the workflow for routine laboratory applications, ensuring high-quality results with minimal reagent consumption. It provides a comprehensive guide from initial sample preparation to high-resolution 3D imaging using the Miltenyi UltraMicroscope Blaze, culminating in final OME-TIFF generation. This version is ideal for researchers requiring rapid processing and high-throughput volumetric analysis of smaller tissue specimens.
Adapted from: Hongcheng Mai et al., "Scalable tissue labeling and clearing of intact human organs," Nature Protocols, 2022. DOI: [https://doi.org/10.1038/s41596-022-00712-8]
Guidelines
The Miltenyi/LaVision Biotech Blaze Ultramicroscope is a lightsheet microscope designed for capturing 3D structures of embryo scale samples. Lightsheet requires samples to be naturally optically transparent or cleared using methods such as BABB, iDisco, Clarity, Scale etc. Blaze has three objectives (1x, 4x, 12x) and four zoom levels (0.6x, 1x, 1.66x, 2.5x) resulting in twelve levels of magnification between 0.6x & 30x.
Materials
- 4% PFA
- PBS
- CHAPS/NMDEA (10% (wt/v) CHAPS and 25% (wt/v) NMDEA in diH2O)
- 1X PBS
- EtOH/H2O solutions (50%, 70%, 100%)
- DCM/MeOH (2:1, v/v)
- diH2O
- Acetic acid solution
- Guanidine hydrochloride (4M)
- Sodium acetate (0.5M)
- Triton X-100
- Goat serum (10% (v/v))
- DMSO (10% (v/v))
- Tween-20 (0.2% (v/v))
- Alexa Fluor® 647 anti-Podoplanin Antibody
- 0.1% Tween-20 in PBS
- Agarose gel/diH2O
- MeOH/H2O solutions (50%, 70%, 100%)
- BABB/MeOH (1:1)
Uterus tissues were fixed with 4% PFA overnight at 4°C and washed with PBS.
Cut the tissue into approximately 5mm x 5mm x 5mm pieces and incubate in 1.8ml of CHAPS/NMDEA solution at 37°C for 2 days.
CHAPS/NMDEA is composed of 10% (wt/v) CHAPS and 25% (wt/v) NMDEA in diH2O. CHAPS (Sigma, #220201), NMDEA (Sigma, #471828).
Wash with 1X PBS at room temperature with rotation for 2 hours X3, then overnight.
Incubate as described below at room temperature under the hood.
Dehydrate with 10ml of 50% EtOH/H2O solution for 1 day.
Dehydrate with 10ml of 70% EtOH/H2O solution for 1 day.
Dehydrate with 10ml of 100% EtOH solution for 1 day.
Delipidate with 10ml of DCM/MeOH (2:1, v/v) solution for 2 days.
Rehydrate with 10ml of 100% EtOH solution for 1 day.
Rehydrate with 10ml of 70% EtOH/H2O solution for 1 day.
Rehydrate with 10ml of 50% EtOH/H2O solution for 1 day.
Rehydrate with 10ml of diH2O solution for 1 day.
Remove the samples from diH2O and place it in 10 ml of acetic acid solution for 1 day at room temperature under the hood.
Wash twice with diH2O for 1 day at room temperature and incubate with 10 ml of guanidine solution for 1 day.
Guanidine solution is composed of 4M guanidine hydrochloride, 0.05M sodium acetate and 2% (wt/vol) Triton X-100 in PBS, pH6.0. Guanidine hydrochloride (Sigma, #3272).
Wash with 1X PBS at room temperature with rotation for 2 hours X3, then overnight.
Block with 10% (v/v) goat serum, 10% (v/v) DMSO and 0.2% (v/v) Triton X-100 in 1X PBS at 4°C on rotator for 3 days.
Incubate with conjugated Ab in the Ab incubation buffer at 4°C on rotator 14 days.
The antibody incubation buffer is composed of 3% (v/v) goat serum, 3% (v/v) DMSO and 0.2% (v/v) Tween-20 in 1X PBS. The conjugated Ab that we used is Alexa Fluor® 647 anti-Podoplanin (Lymphatic Endothelial Marker) Antibody, #916610.
Wash with 0.1% Tween-20 in PBS at room temperature with rotation for 2 hours X3, then overnight.
Embed the tissue in 1% agarose gel/diH2O and cooling for 3 hours.
Gradually dehydrate with MeOH/H2O (v/v) under the hood.
Dehydrate with 10ml of 50% MeOH/H2O solution for 2 hours.
Dehydrate with 10ml of 70% MeOH/H2O solution for 2 hours.
Dehydrate with 10ml of 100% MeOH solution for 2 hours.
Refresh the 100% MeOH solution and then overnight.
Change to BABB/MeOH (1:1) for 5 hours and perform RI matching with BABB solution for 1-4 days until the sample becomes completely transparent.
Tissue Mounting/Imaging Procedure
Place the sample to the imaging chamber. Glue or screw the sample to the chamber.
Mount the chamber on the strip of a lightsheet microscope.
Move the light sheet up or down in xyz until the beam is in the center of the target.
Determine channel and set the objective lens and zoom level. Set the zero position and set scan range from start to end.
Set the file to autosave and press the Start (play) button.
Convert image to Imaris File. Double click the file, select the output location, name the file, and save as format of Imaris*.ims;*.ims. Press the Export data button.