| Product Description | Aladdin's CUBIC-II Solution can be used in conjunction with the CUBIC Animal Tissue Optical Clearing Kit or can be used alone for optical clearing of animal tissues..
Precautions： CUBIC™-II Solution should be thawed completely at room temperature prior to use. Crystals or precipitates present in this product can be dissolved in a 55℃ water bath for a short time until complete dissolution.CUBIC™-II Solution will be ineffective at high temperatures (＞75℃).Do not shake CUBIC™-II Solution vigorously when using to avoid the formation of air bubbles. Large amount of air bubbles present in the solution should be removed prior to use by ultrasonication or vacuum treatment (~0.1 MPa, ~30min) or by leaving at room temperature for at least 7-8 hours.CUBIC™-II Solution is viscous. Please use wide orifice pipette tips to handle it gently and slowly to avoid the formation of air bubbles.After the imaging, samples can be removed from the Mounting Solution, immersed in CUBIC™-II Solution, and stored at room temperature for 1 week. The longer the time in CUBIC™-II Solution, the more transparent the samples. But samples will also swell in the CUBIC™-II Solution. The samples after imaging can also be washed with 1X Wash Buffer, dehydrated with 30% (w/v) Sucrose solution, embedded with O.C.T. compound, then stored at -80℃ for long term storage.CUBIC™-II Solution should be stored in the dark. However, samples can be processed under non-strictly light-proof conditions in order to facilitate the observation of tissue clearing effects.This product is for R&D only. Not for drug, household, or other uses.For your safety and health, please wear a lab coat and disposable gloves during the operation.
Instructions for Use： 1. Sample preparationa. Preparation of reagents and surgical tools.a) Fix solution: -I Solution: Mix -I Solution and distilled water in the ratio of 1:1 and store at room temperature for up to 1 month.b. Preparation of 50% -II Solution: Mix -II Solution and 1X PBS in a 1:1 ratio and store at room temperature for up to 2 weeks.c. Preparation of 1X Wash Buffer: Dilute 100X Wash Buffer with 1X PBS at a ratio of 1:100 and store at room temperature for several months.3. Decolorization and degreasing.a. Remove the fix solution, add 1X Wash Buffer, and wash 3 times at room temperature for 2 hours each with shaking at 60 rpm.b. Add an appropriate amount of 50% -I Solution to immerse the sample completely, and incubate at 37℃ for 4-24 hours with shaking at 60rpm. Gradual transparency of tissue edges will be observed.c. Replace with sufficient new 50% -I Solution to immerse the sample completely for every 4 days. Incubate at 37℃ with shaking at 60rpm until the whole tissue/organ is completely clear.d. Add an appropriate amount of 1X Wash Buffer to immerse the sample completely. Wash at least 3 times at room temperature for 2 hours eachwith shaking at 60 rpm.4. Immunofluorescence staining (optional).a. Incubation with the primary antibody: Dilute the primary antibody with Immunol Staining Primary Antibody Dilution Solution , QuickBlock™ Primary Antibody Dilution Buffer for Immunol Staining or other appropriate dilution buffers at the recommended dilution ratio. Incubate for 3 days at 37℃ on a shaker (60rpm) with appropriate light protection. Note: The dilution ratio can be adjusted appropriately according to the actual staining results.b. Recover the primary antibody and wash 6 times with 1X Wash Buffer for 2 hours each at room temperature.c. Incubation with the secondary antibody: Dilute the secondary antibody with Immnol Fluorescence Staining Secondary Antibody Dilution Solution , QuickBlock™ Secondary Antibody Dilution Buffer for Immunofluorescence or other appropriate dilution buffers at the recommended dilution ratio. Incubate for 3 days at 37℃ on a shaker (60rpm) with appropriate light protection. Note: The dilution ratio can be adjusted appropriately according to the actual staining results.d. Recover the secondary antibody and wash with 1X Wash Buffer at least 3 times for 2 hours each at room temperature. 5. Nucleus Staining (optional).a. Stain samples in 1X Wash Buffer containing 0.5-10 μg/mg DAPI for 2-3 days at 37℃ in the dark with shaking at 60 rpm.Note: The DAPI concentration can be adjusted appropriately according to the actual staining results.b. Wash with 1X Wash Buffer at least 3 times for 2 hours each at room temperature.6. Adjust the refractive index of the sample.a. Add an appropriate amount of 50% -II Solution to immerse the sample completely, place vertically (bubbles tend to form when placed horizontally) and incubate for 6-24 hours at 37℃ with shaking at 60rpm until the sample settles to the bottom.Note: The amount of 50% -II Solution is recommended to be at least 2-3 times the sample volume to ensure complete submersion of the sample during shaking.b. Add an appropriate amount of -II Solution to submerge the sample, place vertically (bubbles tend to form when placed horizontally) and incubate for 24 hours at 37℃ with shaking at 60rpm.Note: The amount of -II Solution is recommended to be at least 2-3 times the sample volume to ensure complete submersion of the sample during shaking.c. Repeat step 6b once.d. Remove the -II Solution completely, add Mounting Solution to submerge the sample for 10 minutes, carefully remove air bubbles from the surface of the sample, and then take photography of the sample. It is recommended to place the sample in a 35mm Confocal Dishe (Φ20mm glass) or on a conventional glass slide for photography.FAQ:1. Is it necessary to use a special type of container for the tissue clearing?No, there is no need to use special types of containers. Common polypropylene, polyethylene or glass vessels can be used. Since animal tissues and organs may swell during the clearing, it is recommended to use slightly larger containers.2. Do the samples swell during the clearing? Does the swelling have any negative impact on the experiment?Samples such as tissues and organs are likely to swell during the clearing. However, this swelling is relatively uniform, and the relative position of the cells remains unchanged. There is no significant negative impact on the experiment except the overall tissue morphology swells.3. Can fresh tissues be cleared without the fixation step?No. Clearing without fixation may lead to changes of cell positions, thus affecting the subsequent immunostaining result. Therefore, it is necessary to fix the sample prior to clearing.4. Can the fixed samples that have been stored for a long time be used for clearing?Yes. This product can be used to clear samples that have been kept in fix solution for several weeks, or samples that have been fixed and stored at -80℃ for several months. However, the immunostaining efficacy may decrease.5. How to estimate the amount of each reagent needed for a sample?For whole bodies of mice, the volume of reagent used must be sufficient to submerge the entire specimen (typically 200-400ml of -I Solution and 100-200ml of -II Solution). For organs, the specimen should be immersed in at least 5 times the volume of the specimen in -I Solution. For example, for a 1cm3 specimen, 20-50ml of -I Solution and 10-30ml of -II Solution are required. The amount of reagent may be appropriately increased when the amount of tissue blood residue is higher, the lipid content is higher, or the tissue is thicker.6. Is it possible to put different tissues/organs or several of the same tissues/organs in one tube for clearing?Several tissues or organs can be cleared together in one tube. For example, heart, lungs, kidneys, spleen, pancreas, and liver can be placed in one tube. However, the stomach and intestines should be separated from other organs and the gastrointestinal contents should be removed as much as possible for clearing.7. Is it necessary to immerse the sample in Mounting Solution for observation after clearing?As -II Solution is an aqueous solution, it may evaporate during observation, making refractive index change, solute deposit, and image acquisition difficult. For short period of observation and photo taking (＜1 hour), samples can be immersed in -II Solution. For longer period of observation (＞1 hour), it is recommended to soak the sample in Mounting Solution.8. Samples are difficult to be observed after clearing.It is recommended to use Light Sheet Fluorescence Microscopy (LSFM) or Confocal Laser Scanning Microscopy (CLSM) to observe transparent samples. In addition, it is recommended not to cut the sample too thin to prevent distortion of the tissue structure due to the nature of the gel. The transparent samples are recommended to be observed in Mounting Solution (Refractive Index/RI = 1.467) with an objective suitable for this RI.9. What is the Refractive Index (RI) of -II Solution?The RI of -II Solution is 1.48-1.49. It is recommended to use an objective lens and immersion oil suitable for this RI. Do not mix the Optical Clearing Reagents in this kit with other solvents (e.g., water) as this may alter the refractive index and affect the observation of samples.10. Why is the sample not fully transparent?a. The pH of the PFA fix solution is too high.A pH greater than 8 may result in excessive fixation and make the sample more difficult to clear. Samples should be fixed in pH 7-7.5. b. Incomplete degreasing.Extend the degreasing time or change fresh -I Solution more frequently. It is recommended to soak the sample in -I Solution at 37℃ for at least 2-5 days and change new solution daily.c. Mismatched refractive index.Make sure that -II Solution is completely dissolved and free of precipitation when using. Try to extend the time and/or replace with new -II Solution.d. Use of old animals.Younger animals are usually better for clearing than older animals.11. How long does the sample need to be degreased?For adult mice, it takes 2-3 days for intestine, pancreas, and spleen, and 1-2 weeks for lung, heart, brain, stomach, muscle, liver, and kidney.12. What kind of tissue staining reagents can be used together with this kit?Large molecule dyes such as immunofluorescence stains, and small molecule dyes, such as nuclear stains (e.g., DAPI, Hoechst, propidium iodide), are compatible with this kit.13. What kind of nuclear stains can be used for cleared samples?We recommend using DAPI , Hoechst 33342 , Hoechst 33258 , Propidium Iodide , etc.14. Is it necessary to use special antibodies for cleared samples?While many proteins do not lose their antigenicity during fixation, degreasing, and decoloring, there are exceptions. It is advisable to properly validate whether an antibody can be used for cleared samples.15. Can fluorescent-labeled secondary antibodies be used?Yes. However, given the time required for antibody incubation, we recommend using fluorescent-labeled primary antibodies to shorten the experimental time.16. What fluorescent proteins are compatible with this kit?This kit is compatible with common fluorescent proteins such as GFP, EGFP, EYFP, mCherry, and mKate2.17. What kind of fluorescent dyes can be used?This kit is compatible with common fluorescent probes such as FITC, Rhodamine, Cy3, Alexa Fluor 488, Alexa Fluor 555, Alexa Fluor 594, and Alexa Fluor 647.Related Products:产品编号产品名称包装C0221APBS500mlC1002DAPI5mg/ml×0.2mlC1006DAPI Staining Solution50mlFCFC020-10pcs 35mm Confocal Dishes (Φ20mm glass)10pcs/bagFCFC020-150pcs 35mm Confocal Dishes (Φ20mm glass)10pcs/bag, 150pcs/boxFS003Scissors (12.5cm, Straight, Sharp)1 setFS007Scissors (12.5cm, Curved, Sharp)1 setFS209Operating Scissors (12.5cm, Straight, Sharp)1 setFS213Operating Scissors (12.5cm, Curved, Sharp)1 setFS225Ophthalmic Forceps (10cm, Straight with hook)1 setFS229Ophthalmic Forceps (10cm, Curved with hook)1 setFS241Hemostatic Forceps (14cm, Straight with Full Tooth)1 setFS245Hemostatic Forceps (14cm, Curved with Full Tooth)1 setFS500Small Animal Surgery Kit1 kitP0098-100mlImmunol Staining Fix Solution100mlP0099-100ML4% Paraformaldehyde Fix Solution, 4% PFA Fix Solution100mlP0103Immunol Staining Primary Antibody Dilution Solution100mlP0108Immnol Fluorescence Staining Secondary Antibody Dilution Solution100mlP0112L Animal Tissue Optical Clearing Kit500mlP0112M Animal Tissue Optical Clearing Kit100mlP0112S Animal Tissue Optical Clearing Kit25mlP0115-100ml-I Solution100mlP0115-500ml-I Solution500mlP0116-100ml-II Solution100mlP0116-500ml-II Solution500mlP0117-100ml 100X Wash Buffer100mlP0118-100ml Mounting Solution100mlP0118-500ml Mounting Solution500mlP0262QuickBlock™ Primary Antibody Dilution Buffer for Immunol Staining100mlP0265QuickBlock™ Secondary Antibody Dilution Buffer for Immunofluorescence100mlST447-1LPBS (1X, premixed powder)1LST448-1LPBS (10X, premixed powder)1L
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