在植物学实验中，用传统的方法不能观察到植物组织的不同层次结构，其原因是：样本中色素对光波的吸收作用和样本各部对光线折射率的不同，造成光线散射，不利于观察。为了更好的观察植物组织内部结构，近二十年来，已有大量组织透明技术被开发应用在了植物组织成像领域，用于观察组织内外部病菌和荧光蛋白。目前常用的植物组织透明方法可以分成基于有机溶剂和基于水性溶剂的透明化方法，其中水合氯醛是最常用的方法，但由于其有特殊气味，对使用者不太友好。
本品采用了基于水性溶剂的透明化方法，在 ClearSee 透明方法的基础上进行了优化和改良，解决了组织褐化问题，适用于轻薄嫩叶组织的整体透明，透明时间适中，一般 3~5 天内可实现完全透明；结构保存相对完整；不太适合用于高度木质化的植物样品。该试剂仅用于科研领域，不适用于临床诊断或其他用途。
自备材料：
1、抽气装置、镊子、显微镜、载玻片
2、多聚甲醛固定液 (4% PFA) 或中性福尔马林固定液 (10% NBF)、1×PBS、蒸馏水、植物组织保存液、稀苯胺蓝染色液、甘油浮载剂、梯度乙醇、二甲苯或环保脱蜡透明液、加拿大树胶。
操作步骤 (仅供参考)：
1、取新鲜组织，置入 4% PFA 或 10% NBF 固定液中，负压抽气使组织沉于底部，固定 12~24h。
2、用镊子将用镊子组织，置于 1×PBS 中浸洗 2 次，每次 2~3min。
3、用镊子取出组织，投入植物组织透明液 (中速) 中室温浸渍透明 3~5d。
4、镊子取出组织用蒸馏水清洗，置于植物组织保存液中保存。
5、(可选) 水洗后放置于载玻片上，滴加稀苯胺蓝染色 1~3h，滴加甘油浮载剂后盖片观察。
6、若需制成永久玻片，应梯度乙醇脱水，二甲苯或脱蜡透明液处理，并用加拿大树胶封固。
注意事项：
1、组织需充分排气固定，否则会影响后续组织透明渗透效果。
2、该试剂对于幼嫩薄叶组织透明效果最佳，成熟组织透明时间较长。
3、透明时间根据组织类型不同可进行适当调整，若想减少透明时间，可以进行真空负压透明。若透明液已明显变色，可更换新的透明液继续透明处理。
4、试剂开封后请尽快使用，以防影响后续实验效果。
5、为了您的安全和健康，请穿实验服并戴一次性手套操作。
In botanical experiments, traditional methods fail to observe multi-layered structures of plant tissues. This is because pigments in specimens absorb light waves and varying refractive indices across different parts of the sample lead to light scattering, which hinders microscopic observation.To better visualize internal structures of plant tissues, numerous tissue clearing techniques have been developed and applied to plant tissue imaging over the past two decades, enabling observation of endophytic and epiphytic pathogens as well as fluorescent proteins.
Common plant tissue clearing approaches fall into two categories: organic solvent-based and aqueous solvent-based methods. Chloral hydrate is the most widely used option, yet it has a pungent odor and poses poor user experience.
This product adopts an aqueous solvent-based clearing strategy optimized and improved on the basis of the ClearSee clearing method to resolve tissue browning. It is suitable for whole-mount clearing of delicate young leaf tissues with a moderate processing period, achieving complete transparency within 3 to 5 days while preserving tissue structure relatively intact. It is not recommended for highly lignified plant samples.This reagent is exclusively for scientific research and must not be used for clinical diagnosis or other purposes.
Materials to Be Prepared by User:
1. Vacuum pumping device, tweezers, microscope, glass slides
2. 4% Paraformaldehyde Fixative (4% PFA) or 10% Neutral Buffered Formalin Fixative (10% NBF), 1×PBS, distilled water, Plant Tissue Preservation Solution, Dilute Aniline Blue Staining Solution, Glycerol Mounting Medium, graded ethanol, xylene or eco-friendly dewaxing clearing solution, Canada Balsam
Operating Procedures (For Reference Only):
1. Collect fresh tissues and immerse them in 4% PFA or 10% NBF fixative. Apply negative pressure to sink the tissues to the bottom and fix for 12–24 hours.
2. Use tweezers to transfer tissues into 1×PBS and rinse twice, 2–3 minutes per wash.
3. Take out the tissues with tweezers and submerge them in Medium-speed Plant Tissue Clearing Solution for room-temperature incubation and clearing over 3–5 days.
4. Retrieve tissues, rinse with distilled water, and store in Plant Tissue Preservation Solution.
5. (Optional) Place rinsed tissues on a glass slide, add dilute aniline blue stain for 1–3 hours, apply glycerol mounting medium and cover with a coverslip for observation.
6. For permanent slide preparation, perform dehydration with graded ethanol followed by treatment with xylene or dewaxing clearing solution, then mount and seal with Canada Balsam.
Precautions:
1. Tissues must undergo thorough degassing and fixation; insufficient pretreatment will impair subsequent penetration and clearing effects.
2. This reagent delivers optimal clearing performance on young and thin leaf tissues, whereas mature tissues require a longer clearing duration.
3. Adjust clearing duration according to tissue type. Vacuum negative pressure treatment can shorten processing time. Replace the clearing solution with a fresh batch if the liquid turns visibly discolored to continue clearing.
4. Use the reagent promptly after opening to avoid compromised experimental results.
5. Wear a lab coat and disposable gloves throughout operation to protect personal safety and health.
ZJ26F0939299
危险品化学品经营许可证（带存储）