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致畸试验骨组织透明试剂盒
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概述
致畸试验骨组织透明试剂盒(Bone Clearing Kit for Teratogenicity Test)由 KOH 溶液、KOH-甘油溶液和试剂甘油溶液组合而成。整套配方严格遵循致畸试验标准配比，全部为即用型液体，无需自行配制稀释。用于食品、化妆品、保健品、化工产品毒理学安全性评价，适配大鼠、小鼠、家兔致畸试验胎仔染色前后梯度透明、脱色、保存全套工序。
作用原理：低浓度氢氧化钾缓慢分解胎仔残留软组织，甘油可缓冲碱性腐蚀、维持骨骼形态，梯度脱去组织内色素与多余染料，避免骨骼软化溶解，实现标本初步褪色透明，衔接后续透明液精制通透处理。
该试剂仅用于科研领域，不适用于临床诊断或其他用途。
操作步骤(仅供参考)：
1、样本预处理(大鼠、小鼠适用)
取每窝 1/2 剖宫活胎仔，二选一处理：
a 剥皮法：小心完整剥皮、去除内脏与全身脂肪，重点清理颈肩区域脂肪组织，操作轻柔避免肋骨、四肢骨断裂；
b 完整胎仔法：胎仔无需剥皮，95%乙醇密封固定 2-3 周，流水冲净残留固定液。
注：家兔致畸试验不可分半取样，全部胎仔均需完整透明染色检查。
2、一级透明脱色(本品直接使用，无需稀释)：
1）将预处理后的胎仔完全浸没于试剂 A(KOH 溶液)中，密封避光室温浸泡 8-72h；
2）定时观察，直至软组织完全透明、骨骼轮廓清晰即可取出，不可长时间浸泡，防止细小骨化中心溶解丢失；
3）透明完毕后流水充分冲洗胎仔表面，洗净残留碱液，随后进行茜素红骨骼染色。
3、分级透明脱色(染色完成后操作)：
1）染色结束后的胎仔浸入试剂 B(KOH-甘油溶液)避光浸泡 1-2d；
2）转入试剂 C(甘油溶液)避光浸泡 2-3d；
3）若软组织紫红色残留较重，可适度延长试剂 B、试剂 C 透明处理时长。
4、镜检观察：
1）两级透明完成后，标本留存于试剂 C 甘油溶液中长期封存，方便镜检与标本留存；
2）采用透射光源，体视显微镜下依次检查颅骨、脊柱、肋骨、胸骨、四肢骨，记录骨化缺失、骨骼融合、肋骨分叉、骨缺损、骨化发育不全等各类骨骼畸形；
3）毒理试验统计要求：所有骨骼畸形、发育异常指标均以窝为单位进行统计，计算总畸胎率、单项骨骼畸形率。
注意事项：
1、透明浸泡阶段务必定时查看标本状态，浸泡超时容易腐蚀细小骨化位点，造成骨骼缺损假阳性结果，干扰畸形判定。
2、用途区分：试剂 A 用于染色前软组织通透；试剂 B、C 用于染色后梯度脱色透明与标本长期保存，不可混用。
3、配套试验方案：另一半胎仔建议使用致畸试验固定液(内脏检查)(T1529295)固定，用于内脏畸形同步检查。
4、为了您的安全和健康，请穿实验服并戴一次性手套操作。
The Bone Clearing Kit for Teratogenicity Test consists of KOH Solution, KOH-Glycerol Solution, and Glycerol Solution. The entire kit is formulated in strict accordance with the standard proportions for teratogenicity testing. All components are ready-to-use liquids and require no further preparation or dilution. It is intended for toxicological safety evaluation of food, cosmetics, health products, and chemical products, and is suitable for the complete workflow of graded clearing, decolorization, and preservation before and after skeletal staining of rat, mouse, and rabbit fetuses in teratogenicity tests.
Principle: Low-concentration potassium hydroxide slowly digests residual soft tissues of the fetuses. Glycerol buffers alkaline corrosion and maintains skeletal morphology. Graded treatment removes tissue pigments and excess stain, prevents softening and dissolution of the skeleton, achieves preliminary decolorization and clearing of the specimens, and facilitates subsequent refined clearing and permeabilization.
This reagent is intended for research use only and is not suitable for clinical diagnosis or other purposes.
Procedure (For Reference Only):
1. Sample Pretreatment (Applicable to Rats and Mice)
Take 1/2 of the live fetuses obtained by cesarean section from each litter and select one of the following two methods:
a Skinning method: Carefully remove the skin completely, remove the internal organs and body fat, with particular attention to removing adipose tissue from the neck and shoulder regions. Handle gently to avoid breakage of the ribs and limb bones;
b Intact fetus method: No skinning is required. Fix the fetuses in 95% ethanol in a sealed container for 2-3 weeks, and rinse thoroughly with running water to remove residual fixative.
Note: For rabbit teratogenicity tests, half-litter sampling is not applicable. All fetuses must undergo complete clearing and staining examination.
2. Primary Clearing and Decolorization (Use directly without dilution):
1) Completely immerse the pretreated fetuses in Reagent A (KOH Solution), seal and incubate at room temperature protected from light for 8-72 h;
2) Check regularly. Remove the fetuses once the soft tissues are completely transparent and the skeletal outlines are clearly visible. Do not soak for an excessively long time to prevent dissolution and loss of small ossification centers;
3) After clearing is completed, thoroughly rinse the surface of the fetuses with running water to remove residual alkaline solution, and then proceed with Alizarin Red skeletal staining.
3. Graded Clearing and Decolorization (Performed after staining):
1) Immerse the stained fetuses in Reagent B (KOH-Glycerol Solution) and incubate protected from light for 1-2 d;
2) Transfer to Reagent C (Glycerol Solution) and incubate protected from light for 2-3 d;
3) If substantial purple-red staining remains in the soft tissues, the clearing treatment time with Reagent B and Reagent C may be appropriately extended.
4. Microscopic Examination:
1) After the two-stage clearing procedure is completed, store the specimens in Reagent C (Glycerol Solution) for long-term preservation, facilitating microscopic examination and specimen storage;
2) Using transmitted light, examine the skull, vertebral column, ribs, sternum, and limb bones sequentially under a stereomicroscope, and record various skeletal abnormalities, including absent ossification, skeletal fusion, bifurcated ribs, bone defects, and incomplete ossification;
3) Statistical requirements for toxicological testing: All skeletal malformations and developmental abnormalities shall be statistically analyzed on a litter basis, and the total fetal malformation rate and individual skeletal malformation rates shall be calculated.
Precautions:
1. During the clearing and soaking stage, the condition of the specimens must be checked regularly. Excessive soaking may corrode small ossification sites, causing false-positive skeletal defect results and interfering with malformation assessment.
2. Intended use of reagents: Reagent A is used for soft tissue permeabilization before staining; Reagents B and C are used for graded decolorization and clearing after staining and for long-term specimen preservation. Do not mix or interchange the reagents.
3. Supporting test protocol: The other half of the fetuses is recommended to be fixed using Teratogenicity Test Fixative (Visceral Examination) (T1529295) for simultaneous examination of visceral malformations.
4. For your safety and health, wear a laboratory coat and disposable gloves during operation.
规格
别名
致畸试验骨组织透明液
英文别名
Bone Tissue Clearing Solution for Teratogenicity Testing
规格或纯度
BioReagent, 即用型, 组织学级, 用于显微镜
英文名称
Bone Clearing Kit for Teratogenicity Test
应用
Fetal Bone Clearing
样品类型
胎仔
检测物种
大鼠、小鼠、兔
检测仪器
显微镜
储存与运输
储存条件
室温
运输条件
常规运输
稳定性与储存
Store at room temperature long term (12 months).
组分与储存
组分货号 | 组分名称 | 物理外观 | 3×500mL | 储存 |
B1529988A | 试剂(A)：KOH溶液 | 液体 | 500mL | 室温 |
B1529988B | 试剂(B)：KOH-甘油溶液 | 液体 | 500mL | 室温 |
B1529988C | 试剂(C)：甘油溶液 | 液体 | 500mL | 室温 |
高级数据
质检证书(CoA,COO,BSE/TSE 和分析图谱)
C of A & Other Certificates(BSE/TSE, COO):
输入批号以搜索分析图谱:
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