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.
