| 货号 (SKU) | 包装规格 | 是否现货 | 价格 | 数量 |
|---|---|---|---|---|
| C743121-100ml |
100ml |
期货  |
| |
| C743121-500ml |
500ml |
期货 |
|
| 稳定性与储存 | -20℃避光保存，至少一年有效。 |
|---|---|
| 英文名称 | CUBIC-II Solution |
| 储存温度 | 避光,-20°C储存 |
| 运输条件 | 超低温冰袋运输 |
| 产品介绍 |
阿拉丁研发生产的CUBIC-II Solution，即CUBIC动物组织折光率调节试剂，可以和阿拉丁生产的CUBIC Animal Tissue Optical Clearing Kit配套使用，也可以单独用于透明化处理后组织器官等样品折光率的调节。 -20℃避光保存，至少一年有效。CUBIC-II Solution使用前，需室温静置至完全融化，确保液体透明并且没有沉淀。如果有晶体析出或溶液不透明，可以短时间置于55℃水浴溶解至完全透明并且没有沉淀。CUBIC-II Solution在高温(＞75℃)下失效，注意不可高温处理。CUBIC-II Solution使用时请勿剧烈摇晃，避免产生气泡。如果不慎导致CUBIC-II Solution中产生大量气泡，请采用超声波清洗机处理或抽真空处理(~0.1 MPa, ~30min)以去除气泡，或室温静置(至少7-8小时)使气泡自行消除，以免导致组织器官周围或内部出现气泡。CUBIC-II Solution较粘稠，使用时请使用大口枪头(可自行用剪刀剪去枪头尖端)缓慢吸取，并缓慢加入至样品中，请勿剧烈吹打，避免产生大量气泡。完成照片和视频拍摄后的组织器官样品可以去除Mounting Solution，然后可以浸泡在CUBIC-II Solution中室温保存1周。时间越长，样品透明度越高，但也会导致组织膨胀。完成照片和视频拍摄后的样品可以采用1X Wash Buffer清洗，再用30% (w/v)蔗糖溶液脱水，然后用O.C.T化合物包埋，并在 使用说明： 1.组织样品的制备。a.试剂和器材的准备。a)固定液：推荐使用阿拉丁生产的4%多聚甲醛固定液或免疫染色固定液。b)麻醉剂：根据实验需求选择适合麻醉剂，如戊巴比妥钠(Sodium pentobarbital)或氯胺酮(Ketamine)等。c)灌注液：1X PBS/肝素钠(10U/ml)溶液或1X PBS、4%多聚甲醛固定液。d)器材：推荐使用阿拉丁生产的实验手术剪、眼科镊 、止血钳、输液器的针头(一般小鼠建议采用6或7号针头，大鼠建议采用10至12号针头)。b.动物麻醉。例如1%戊巴比妥钠(建议按体重计算麻药的使用量：80ml/kg)进行腹腔注射麻醉小鼠。c.沿肋骨下方的体表和腹壁横切口，快速剪开隔膜，抬起胸骨，暴露心脏。d.从心尖部开始由左下方斜向右上方插针直至升主动脉内，若遇阻力，可尝试改变方向重试，切勿蛮力插入。e.用止血钳夹住心室(或手持)，固定针头，防止泄漏，右心耳剪小口。f.灌注1X PBS/肝素钠(10U/ml)，灌注速度先慢后快，冲洗血管，去除血液。一般以从右心耳流出的液体清亮、肝脏呈灰白色为标准。g.灌注4%多聚甲醛固定液进行固定，灌注速度先快后慢，以肌肉变硬为标准，一般用量约相当于实验动物体重。也可以解剖出所需的组织器官后固定。h.解剖出所需组织器官，浸泡在4%多聚甲醛固定液中，在摇床上固定过夜。2.透明化试剂的准备。a.50% -I Solution的配制：将-I Solution和蒸馏水按1:1的比例混匀，室温下可保存1个月。b.50% -II Solution的配制：将-II Solution和1X PBS按1:1的比例混匀，室温下可保存2周。c.1X Wash Buffer的配制：将100X Wash Buffer和1X PBS按1:100比例稀释，室温下可保存数月。3.组织器官等的脱脂脱色。a.弃固定液，加1X Wash Buffer，室温摇床(60rpm)洗涤3次，每次2小时。b.加入适量50% -I Solution浸没样品，37℃摇床(60rpm)孵育4-24小时。可观察到组织边缘逐渐透明。(注：50% -I Solution使用量可根据样品体积适当调整，确保样品在摇晃过程中完全浸没即可)。c.加入适量的-I Solution浸没样品，37℃摇床(60rpm)孵育，每4天更换一次新鲜溶液，直至组织器官样品完全透明。(注：-I Solution使用量可根据样品体积适当调整，确保样品在摇晃过程中完全浸没即可)。d.加入适量1X Wash Buffer浸没样品，室温摇床(60rpm)至少洗涤3次，每次2小时。4.免疫荧光染色(选做)。a.一抗孵育：用免疫染色一抗稀释液、QuickBlock™免疫染色一抗稀释液或其它适当的一抗稀释液按照一抗推荐的稀释比例进行稀释。37℃摇床(60rpm)适当避光孵育3天。注：一抗稀释比例可以根据实际染色效果进行适当调整。b.回收一抗，用1X Wash Buffer，室温洗涤6次，每次2小时。 c.二抗孵育：用免疫荧光染色二抗稀释液、QuickBlock™免疫荧光染色二抗稀释液或其它适当的二抗稀释液按照二抗推荐的稀释比例进行稀释。37℃摇床(60rpm)避光孵育3天。注：二抗稀释比例可以根据实际染色效果进行适当调整。d.回收二抗，用1X Wash Buffer，室温洗涤至少3次，每次2小时。 5.细胞核染色(选做)。a.DAPI孵育：1X Wash Buffer稀释DAPI至0.5-10μg/mg工作液，37℃摇床(60rpm)避光孵育2-3天。注：稀释比例可以根据实际染色效果进行适当调整。b.用1X Wash Buffer，室温至少洗涤3次，每次2小时。6.调节样品折光率。a.加入适量50% -II Solution浸没样品，竖立放置(水平放置时易产生气泡)，37℃水平摇床(60rpm)孵育6-24小时，直至样品沉底。注：50% -II Solution使用量可根据样品体积适当调整；建议每次使用量至少为样品体积的2-3倍，确保样品在摇晃过程中完全浸没。b.加入适量-II Solution浸没样品，竖立放置(水平放置时易产生气泡)，37℃水平摇床(60rpm)孵育24小时。注：-II Solution使用量可根据样品体积适当调整；建议每次使用量至少为样品体积的2-3倍，确保样品在摇晃过程中完全浸没。c.重复步骤6b一次。d.充分去除-II Solution，加入Mounting Solution浸没样品10分钟，小心剔除样品表面气泡后即可用于拍照。推荐将浸泡样品放置在 35mm玻底共聚焦培养皿(玻璃直径20mm) 中或常规载玻片上进行拍照。常见问题：1.动物组织透明化过程中是否需要使用特殊类型的容器?无须使用特殊类型的容器。可以使用实验室常用的聚丙烯或聚乙烯类塑料器皿或玻璃器皿。由于动物组织器官在透明化过程中可能会膨胀，建议使用稍大一点的容器。2.样本在透明化过程中会膨胀吗？膨胀对实验有什么负面影响吗?组织器官等样品在透明化过程中很可能会膨胀。但这种膨胀是接近线性并且比较均匀的，细胞相对位置是保持不变的。除了整体形态会膨胀外，其它不会产生很明显的负面影响。3.新鲜组织可以跳过固定步骤直接进行透明化吗?不可以。在未进行固定的情况下进行组织透明化，可能导致细胞相对位置紊乱，也可能影响后续免疫染色等的效果。因此，在透明化之前需要先固定样本。4.是否可以使用长时间保存的固定样品进行透明化处理?可以的。本产品可以用于已经在固定溶液中浸泡固定数周的样品，以及已经被固定并在-80℃存储数月的样品。但如果计划对样品进行免疫染色，长期保存的固定样品可能会出现目标蛋白免疫染色效果下降的情况。5.如何粗略估计样本需要每种试剂的用量？对于小鼠全身透明化，所用试剂的体积必须足以浸没整个标本(一般需要200-400ml的-I Solution和100-200ml的-II Solution)。对于器官透明化来说，样本应浸泡在至少5倍于样本体积的-I Solution中。例如，对于1cm3的标本，总共需要20-50ml的-I Solution和10-30ml的-II Solution。当组织血残留量较多、脂质含量较高或组织较厚时，透明化所需要的试剂总量可能会适当增加。6.能否把不同的组织器官或几个相同的组织器官放在一个管子进行透明化？几个组织器官可以在一个管子里处理。例如，心、肺、肾、脾、胰腺和一块肝脏可放在同一个管子，心、肺、肾、脾、胰腺等几个适当的小器官也可放在同一个管子。但胃和肠应该被分开到不同的管子里，并且应尽可能地清除胃肠道内容物。7.透明化后样品观察时是否一定需要浸泡在Mounting Solution中?由于-II Solution是水性溶液，在较长时间的观察过程中，可能会蒸发，导致折光率变化和溶质沉积，造成图像采集困难。对于短时间观察和拍照(＜1小时)，可以在-II Solution中浸泡样品。对于较长的观察时间(＞1小时)，建议使用Mounting Solution浸泡样品。8.透明化后样品观察困难。建议使用光片荧光显微镜(LSFM)或激光共聚焦扫描显微镜(CLSM)观察透明化的样品。此外，建议不要将需透明化处理的样品切割太薄，防止凝胶性质导致组织结构扭曲。透明化后的样品推荐在Mounting Solution (Refractive Index/RI = 1.467)中进行拍照观察，并需要使用适合这些RI的物镜进行观察。9.-II Solution的折射率(Refractive Index, RI)是多少?-II Solution的RI为1.48-1.49。建议使用适合这些折光率的物镜和浸没油。请勿将Optical Clearing Reagents试剂与其它溶剂(如水)混合，以免改变其折光率，影响透明化样品的观察。10.为什么样本不能充分透明?a.PFA固定液pH过高。pH值大于8可能导致过度固定，使样品更难透明。建议试着将pH值调整到7-7.5之间。b.脱脂不完全。尝试延长脱脂时间或更加频繁地更换新鲜的-I Solution。建议将样品在37℃的-I Solution中浸泡至少2-5天，每天更换新鲜试剂。c.折光率不匹配。使用时请确保-II Solution溶解完全且没有沉淀。尝试延长时间和/或更换新鲜的-II Solution。d.使用老龄动物。年轻的动物透明化效果通常优于年老的动物。11.样品需要脱脂处理多久？对成年小鼠，肠、胰腺和脾脏大约需要2-3天，肺、心脏、大脑、胃、肌肉、肝脏和肾脏大约需要1-2周。12.什么样的组织染色试剂可以配合透明化试剂盒一起使用?例如免疫荧光染色等大分子染色、细胞核荧光染色(例如DAPI、Hoechst、碘化丙啶)等小分子染色都是可以和透明化的样品兼容的。13.透明化样品可以使用什么样的核染色试剂?推荐使用DAPI 、Hoechst 33342染色液、Hoechst 33258染色液、Propidium Iodide 等。14.是否需要购买特殊的抗体用于透明化处理后的样品?虽然许多蛋白质在固定、脂肪清除和脱色过程中不会失去其抗原性，但也有例外。建议最好对所使用的抗体进行适当验证。15.可以使用荧光标记的二抗吗?可以。但考虑到样品中每个抗体孵育所需的时间，更建议使用荧光标记一抗，以缩短实验周期。16.可以兼容哪些荧光蛋白?本产品具有较好的荧光蛋白兼容性，可以兼容GFP、EGFP、EYFP、mCherry和mKate2等常见荧光蛋白。 17.可以使用什么样的荧光染料?FITC、Rhodamine、Cy3、Alexa Fluor 488、Alexa Fluor 555、Alexa Fluor 594、Alexa Fluor 647等常见荧光探针都是可以的。 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. |
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