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致畸试验骨组织透明液B (甘油溶液)
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概述
致畸试验骨组织透明液B (甘油溶液)是致畸试验骨组织透明液套装（Bone Clearing Kit for Teratogenicity Test）的组成部分之一，与KOH溶液、KOH-甘油溶液配合使用。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，适用于大鼠、小鼠、家兔致畸试验胎仔染色前后的梯度透明、脱色及保存等工序。
作用原理：低浓度氢氧化钾可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，通过梯度处理逐步脱去组织内色素及多余染料，避免骨骼软化溶解，实现标本进一步透明处理，并便于后续观察及保存。
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：
1)将预处理后的胎仔完全浸没于致畸试验骨组织透明液(KOH溶液)(B1529929)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致畸试验骨组织透明液 A(KOH-甘油溶液)(B1529923)避光浸泡 1-2d；
2)转入致畸试验骨组织透明液B(甘油溶液)(B1529922)避光浸泡 2-3d；
3)若软组织紫红色残留较重，可适度延长 KOH-甘油溶液、甘油溶液透明处理时长。
4、镜检观察：
1)两级透明完成后，标本留存于致畸试验骨组织透明液B(甘油溶液)(B1529922)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：KOH 溶液用于染色前软组织通透；KOH-甘油溶液、甘油溶液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。
Bone Tissue Clearing Solution B (Glycerol Solution) for Teratogenicity Testing is one of the components of the Bone Clearing Kit for Teratogenicity Test and is intended for use in combination with KOH Solution and KOH-Glycerol Solution. The complete formulation is prepared according to the standard proportions used in teratogenicity testing. All components are supplied as ready-to-use liquids and require no further preparation or dilution. The product is intended for toxicological safety evaluation of foods, cosmetics, health products, and chemical products, and is suitable for the graded clearing, decolorization, and preservation of rat, mouse, and rabbit fetuses before and after skeletal staining in teratogenicity studies.
Principle: Low-concentration potassium hydroxide gradually digests residual fetal soft tissues, while glycerol helps buffer alkaline corrosion and maintain skeletal morphology. Stepwise treatment progressively removes tissue pigments and excess stain, reduces the risk of skeletal softening or dissolution, and enables further specimen clearing for subsequent observation and long-term preservation.
This reagent is intended for research use only and is not suitable for clinical diagnosis or other purposes.
Operating Procedure (For Reference Only):
1. Sample Pretreatment (Applicable to Rats and Mice)
Select 1/2 of the live fetuses delivered by cesarean section from each litter and process using 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 adipose tissue in the neck and shoulder regions. Handle gently to avoid fractures of the ribs and limb bones;
b. Intact fetus method: The fetuses do not need to be skinned. Fix them in 95% ethanol in a sealed container for 2–3 weeks, then rinse thoroughly under running water to remove residual fixative.
Note: For rabbit teratogenicity tests, half-litter sampling is not applicable. All fetuses should undergo complete clearing and staining examination.
2. Primary Clearing and Decolorization:
1) Completely immerse the pretreated fetuses in Bone Tissue Clearing Solution (KOH Solution) for Teratogenicity Testing (B1529929), seal the container, and incubate at room temperature protected from light for 8–72 h;
2) Observe the specimens at regular intervals. Remove them once the soft tissues are completely transparent and the skeletal contours are clearly visible. Avoid prolonged immersion to prevent dissolution and loss of small ossification centers;
3) After clearing is complete, rinse the fetal surface thoroughly under running water to remove residual alkaline solution, and then proceed with Alizarin Red skeletal staining.
3. Graded Clearing and Decolorization (After Staining):
1) After staining, immerse the fetuses in Bone Tissue Clearing Solution A (KOH-Glycerol Solution) for Teratogenicity Testing (B1529923) and incubate protected from light for 1–2 d;
2) Transfer the fetuses to Bone Tissue Clearing Solution B (Glycerol Solution) for Teratogenicity Testing (B1529922) and incubate protected from light for 2–3 d;
3) If substantial purplish-red coloration remains in the soft tissues, appropriately extend the clearing time in the KOH-glycerol solution and glycerol solution.
4. Microscopic Examination:
1) After completion of the two-step clearing process, store the specimens long-term in Bone Tissue Clearing Solution B (Glycerol Solution) for Teratogenicity Testing (B1529922) to facilitate microscopic examination and specimen preservation;
2) Using transmitted light, examine the skull, vertebral column, ribs, sternum, and limb bones sequentially under a stereomicroscope, and record skeletal abnormalities including absent ossification, skeletal fusion, bifurcated ribs, bone defects, and incomplete ossification;
3) Toxicological test statistics: All skeletal malformations and developmental abnormalities should be analyzed on a litter basis, and the total fetal malformation rate and individual skeletal malformation rates should be calculated.
Precautions:
1. During the clearing and immersion stages, the condition of the specimens must be checked regularly. Excessive immersion may corrode small ossification sites, resulting in false-positive bone defects and interfering with malformation assessment.
2. Distinction of use: KOH solution is used for soft-tissue clearing before staining; KOH-glycerol solution and glycerol solution are used for graded decolorization and clearing after staining and for long-term specimen preservation. These solutions must not be used interchangeably.
3. Supporting test protocol: The other half of the fetuses are recommended to be fixed with 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
规格或纯度
BioReagent, 即用型, 组织学级
稳定性与储存
Store at room temperature long term (12 months).
英文名称
Bone Tissue Clearing Solution B (Glycerol Solution) for Teratogenicity Testing
储存条件
室温
运输条件
常规运输
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生物产品
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生物试剂/缓冲液
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