Ce3D™ Tissue Clearing Kit

Instructions for Use

 

The Ce3D™ (clearing-enhanced 3D) Tissue Clearing Kit works by simple immersion of tissue into the clearing reagent and does not require special equipment or containers.

 

Use caution and the appropriate PPE when working with clarifying reagents. Refer to the MSDS for full details. 

 

Materials Provided

 

Test Size		Cat. No.				Ce3D™ Tissue Clearing Solution				Ce3D™ Permeabilization/Blocking Buffer				Ce3D™ Antibody Diluent Buffer			Ce3D™ Wash Buffer
10 tests		427701				5 mL				15 mL				15 mL			50 mL
50 tests		427702				25 mL				50 mL				50 mL			250 mL

 

Materials Required (not supplied)

 

• HyClone™ PBS (1X) w/o calcium and magnesium
• Heparin
• Fixation buffer
• High resolution agarose
• Slides and cover glasses
• 24-well plate
• Spacer

 

Note: This protocol is optimized for staining with samples with 500 µm thickness. For samples with a different thickness, the end user may need to perform further testing. Use 1% PFA if best clearing effect is desired, or 4% PFA for optimal epitope preservation and immunostaining. For additional support, please contact us at: biolegend.com/en-us/contact-technical-service

 

Ce3D™ Tissue Clearing Workflow

 

 

Table 1. Whole Animal Perfusion and Tissue Sectioning Protocol

 

Workflow	Step		Instruction				Whole Animal (Mouse)								Notes				Time
Whole Animal Perfusion	1		Prepare fresh heparin/ PBS solution at the indicated dosage.				Add 10 units of heparin per mL of PBS. Use ≥ 50 mL per mouse. Keep chilled at 4°C.								It is recommended to prepare fresh heparin/ PBS solution. !     Steps 1-7 and step 10 must be carried out using chilled buffers.				~ 2.5 hrs for steps 1-5
	2		Prepare fresh PFA at the desired volume and concentration.				Use BioLegend's fixation buffer (4% PFA solution). Dilute to 1% in PBS if needed. Alternatively, dilute 16% PFA in PBS to a 1% or 4% solution. Use ≥ 50 mL per mouse. Keep chilled at 4°C.								PFA is light-sensitive and should be protected from light or stored in the dark.
	3		Perform transcardial perfusion wash.				Perform perfusion in the chemical fume hood under anesthesia. Perfuse with ≥ 25 mL of chilled heparin/PBS solution at a pump speed of 0.5 mL per minute.								This step is to wash out blood containing sources of autofluorescence such as hemoglobin from organs. To avoid blood clotting and over-fixation, we recommend perfusion on a chilled pad throughout the process.
	4		Perform transcardial perfusion fixation.				Perfuse with ≥ 25 mL of 1% or 4% PFA solution at 4°C at a pump speed of 0.5 mL per minute.								Perfuse slowly on a chilled pad. Incomplete fixation or over-fixation will affect immunostaining or clearing efficiency.
	5		Harvest organ.				Place harvested organ in a 50 mL conical tube. Keep tube chilled. Wash with ≥ 25 mL heparin/PBS solution at 4°C until the solution is cleared of blood.								Multiple organs can be placed in a 50 mL conical tube.
Organ Fixation	6		Perform organ fixation.				Fix organ with ≥ 25 mL of 1% or 4% PFA at 4°C for 24 hours. Use the same percentage of PFA as in step 4.								Replace PFA with freshly prepared solution if the solution becomes cloudy or pink to ensure complete removal of blood and efficient fixation.				24 hrs
Organ Wash	7		Perform PBS wash.				Wash organ in PBS at 4°C for 1 hour. Repeat wash two additional times.								Residual PFA may lead to over-fixation and affect immunostaining. The fixed organ can be store at 4°C in a buffer containing 0.05% sodium azide in PBS for 2 weeks.				3 hrs
Agarose embedding	8		Prepare 2% agarose solution in PBS.				Measure the appropriate amount of solid agarose and PBS. Melt the agarose in a beaker containing PBS in the microwave with occasional stirring until the agarose is completely dissolved. Keep the solution on a 65°C hot plate to stop agarose from solidifying.								Volume per organ depends on the container used for embedding.
	9		Embed fixed organ in 2% agarose.				Stir the agarose before pouring into a small plastic weight boat. The recommended temperature for agarose is 40-42°C. Transfer the organ using a spatula. Use a Kimwipe and gently wipe around the organ to remove excess PBS. If a specific orientation is required, use a small amount of 2% agarose solution to first make an agarose bed. Then, place the tissue on the agarose bed and fill the container until the organ is completely immersed in agarose. Wait until the agarose has solidified before tissue sectioning.								Do not embed in hot agarose. This may cause tissue autofluorescence. To accelerate agarose solidification, the organ-agarose block can be transferred to a humidity chamber to keep the block hydrated. Cover the container and place at 4°C for 30-60 minutes. The tissue block can be stored at 4°C overnight in a well-hydrated container.
Tissue Sectioning	10		Prepare tissue sections using a vibratome.				Cut 500 μm sections at 4°C. The suggested parameters for PFA-fixed mouse tissue are speed at 0.3 mm/s and amplitude at 1.0 mm. Transfer tissue sections to a 24-well plate.								Tissue sections can be stored at 4°C in a buffer containing 0.05% sodium azide in PBS for up to two weeks.				4 hrs

 

Table 2. Immunostaining and Tissue Clearing Protocol

 

Multicolor Tissue Staining				Step	Instruction			500 μm thick tissue (see Note on side 1 for different thicknesses)							Notes				Time
Tissue Section Permeabilization and Blocking				1	Permeabilize and block tissue section.			Move tissue section to a 24-well plate. Handle with care to avoid tissue damage. Use 500 µL of Ce3D™ Permeabilization/Blocking Buffer per well per tissue section. Incubate at RT with gentle shaking for 2 days.							Replace with fresh Ce3D™ Permeabilization/ Blocking Buffer if the solution becomes cloudy or pink. Tissue section can be stored in this buffer at 4°C for up to one week.				2 days
Antibody Cocktail Preparation				2	Prepare antibody dilution at the desired concentration.			Discard the Permeabilization/Blocking Buffer and replace with antibody cocktail prepared in Ce3D™ Antibody Diluent Buffer. Use 500 µL per well per tissue section. Incubate at RT with gentle shaking for 2 days.							Centrifuge the diluted antibody solution at 13000 rpm for 10 minutes to remove antibody aggregates. Tissue sections can be stored at 4°C in this buffer for up to 1 week. For BioLegend- validated antibodies, we recommend the concentration range listed for 2D IHC.				2 days
Tissue Section Washing				3	Wash tissue section.			Discard the antibody cocktail solution. Add 500 μL of Ce3D™ Wash Buffer per well per tissue section. Incubate at RT with gentle shaking for ~8 hours. Discard the wash buffer and repeat this step twice more. At the second washing step, a counterstaining dye, such as DAPI, can be added to the wash buffer.							Tissue sections can be stored at 4°C in Ce3D™ Wash Buffer for up to one week. Tissues can be washed up to ten times if extra washes are desired.				1 day
Tissue Section Clearing				4	Clear tissue section.			Remove wash buffer completely as excess buffer may interfere with efficient tissue clearing. Add 500 μL of Ce3D™ Tissue Clearing Solution per well per tissue section. Incubate at RT with gentle shaking for ~2 -12 hours. Refer to Table 3 for suggested tissue clearing times.							Ce3D™ Tissue Clearing Solution is viscous. Gently mix before use.				~2-12 hrs
Mounting Tissue Section on Slide				5	Prepare sample chamber.			Assemble the sample chamber by attaching a spacer onto a slide. Fill the space with Ce3D™ Tissue Clearing Solution. Carefully transfer and place the tissue section in the solution and cover with a coverslip. Avoid air bubbles. Use nail polish to seal the edges of the coverslip. Air-dry for 2 hours. Slides can be stored in the dark at 4°C for 2 weeks.							Choose appropriate spacer thickness.				~2 hrs
Reverse Tissue Clearing				6	(Optional) Reverse tissue clearing.			If 2D IHC or other downstream application is desired, remove the tissue section from the imaging chamber and wash in PBS at RT for 30 minutes to reverse the clearing effect.

 

Table 3. Recommended Tissue Clearing Time for Different Organs

 

Time													Organ
2 hours													Intestine, lymph node
4 hours													Spleen, thymus, lung
> 8 hours (overnight)													Kidney, liver, testis, brain

 

Table 4. Troubleshooting Guidelines

 

Problem								Possible cause					Recommended solution
Organ contains blood								Inefficient heparin/PBS perfusion wash					Increase heparin/PBS units/mL. Ensure that heparin is not expired. Chill the whole mouse at 4°C throughout the perfusion.
								Perfusion was carried out too fast					Perfuse with chilled heparin/PBS solution at a pump speed of 0.5 mL per minute.
Not enough fixation								Expired or old fixative solution					Use freshly prepared fixative solution. PFA is light-sensitive and should be protected from light and stored in the dark.
								Insufficient fixation					Perfuse with at least 25 mL of chilled fixative solution per mouse.
													Ensure the needle is properly inserted into the left ventricle throughout perfusion.
Over-fixation								Fixation temperature is too high					Use chilled fixative buffer and perform cardiac perfusion on a chilled pad. Avoid working under a heat source such as light bulb.
Agarose not adhering to tissue								Wet organ surface when embedding in agarose					Ensure the surface of the organ is dried using Kimwipe before embedding into melted agarose. Chill buffers and cutting instruments. Alternatively, prepare 3% agarose.
Dried tissue								Insufficient permeabilization and blocking					Increase tissue permeabilization and blocking time.
Uneven, strong staining on the surface of tissue								High antibody concentration					Reduce antibody concentration.
								Insufficient permeabilization and blocking					Increase tissue permeabilization and blocking time.
Uneven or weak staining								Insufficient immunolabeling					Antibody concentration may be too low. Use lower antibody dilution and mix well before use.
													Increase antibody staining incubation time.
								High background					Increase tissue permeabilization and blocking time. Avoid over-fixation.
								Insufficient fixation					Use 4% PFA instead of 1% PFA to increase tissue fixation.
								Fat or tissue residue affecting tissue staining and imaging					Remove residual tissue before immunostaining.
High background signal								Insufficient tissue blocking					Increase tissue permeabilization and blocking time.
								Autofluorescence from blood					Increase cardiac perfusion wash and tissue permeabilization/block time to remove blood.
Strong fluorescent spots in the image								Antibody aggregation					Centrifuge diluted antibody solution at 13000 rpm for 10 minutes.
								Buffer contamination					Prepare fresh antibody dilutions.
Insufficient tissue clearing								Strong or over-fixation					Reduce fixation time. Chill buffer and animal throughout perfusion.
													Perfuse and fix on ice and with chilled buffers.
													Use 1% PFA instead of 4% PFA as fixative solution.
								Insufficient permeabilization					Increase tissue permeabilization and blocking time.
								Insufficient Ce3D™ Tissue Clearing Solution					Increase the amount of Ce3D™ Tissue Clearing Solution.
													Remove wash buffer as much as possible.
								Excess wash buffer interfering with refractive index of Ce3D™ Tissue Clearing Solution					Perform tissue clearing twice.
													Use fresh Ce3D™ Tissue Clearing Solution to seal in the sample chamber.
													DO NOT REUSE Ce3D™ Tissue Clearing Solution!
								Altered refractive index					Tighten the cap of the Ce3D™ Tissue Clearing Solution and seal with parafilm after each use. Store at 4 °C.
Air bubbles in the sample chamber or Ce3D™ Tissue Clearing Solution								Vigorous vortexing of Ce3D™ Tissue Clearing Solution					Gently mix Ce3D™ Tissue Clearing Solution 1 hour prior use. Let solution sit for 1 hour to eliminate the air bubbles.
								Air bubbles on or in the tissue					Place the tissue in PBS and gently shake until bubbles disappear.
								Air bubbles formed during coverslip sealing					Use clean tweezers or forceps to remove bubbles.
Tissue was not labeled with antibody								Antibody is not compatible with 3D IHC					Use antibodies validated for IHC and immunofluorescence staining.
													Over-fixation affecting access to antibody epitopes.
								Poor antibody labeling					Antigen retrieval may be necessary to increase tissue permeability and antibody access to antigen.