Under normal conditions, the basic proteins (nuclear proteins) bound to sperm nucleus DNA undergo a natural maturation process from histones to protamines. The mature protamines provide special protection for sperm genes (DNA). The gradual replacement of histones by protamines is referred to as sperm nucleoprotein transition, which has important physiological significance. The sperm nucleus carries all the paternal genetic information, and these genes can only be expressed after fertilization. Before fertilization, sperm genes are tightly condensed under the special protection of protamines, with no DNA transcription. However, abnormal nucleoprotein transition can lead to male infertility or early embryonic death and miscarriage. The mechanisms are as follows: ① Sperm DNA becomes unstable and susceptible to damage, making conception difficult; ② Once fertilized, the sperm nucleus cannot decondense normally due to abnormal nucleoprotein transition, thus interfering with the fusion of male and female pronuclei; ③ The embryo fails to develop normally, resulting in embryonic death and miscarriage. Therefore, the amount of histones serves as an important indicator of sperm maturity.
Sperm Nucleoprotein Staining Solution (Eosin‑Aniline Blue Method). The principle is that under acidic conditions, aniline blue can specifically bind to lysine residues rich in histones in the sperm nucleus to form a violet‑blue compound. The maturity of spermatozoa is judged according to the staining intensity. This product is for research use only and not intended for clinical diagnosis or other applications.
Product Components and Storage Conditions:
S1511569
| Component
| 20T
| Storage
|
S1511569A
| 10× Wash Buffer Concentrate
| 10mL
| RT
|
S1511569B
| Fixative Solution
| 10mL
| 2-8℃. Store in the dark.
|
S1511569C
| Aniline Blue Stain
| 10mL
| RT
|
S1511569D
| 10× Elution Buffer Concentrate
| 10mL
| RT
|
S1511569E
| Eosin Counterstain
| 10mL
| RT |
Materials Required:
1. Distilled water, fresh semen sample, graded ethanol, xylene or eco-friendly dewaxing solution, eco-friendly clearing solution, neutral balsam
2. EP tubes, pipettes or pipettors, centrifuge, incubator, adhesive microscope slides
Protocol (For Reference Only):
1. Prepare working wash solution: Dilute the 10× Wash Buffer Concentrate with distilled water at a ratio of 1:10.
2. Prepare working elution solution: Dilute the 10× Elution Buffer Concentrate with distilled water at a ratio of 1:10.
3. Transfer 0.2–0.5 mL of liquefied semen into an EP tube, add 1–1.5 mL working wash solution, and mix by repeated pipetting. Centrifuge at 2000 rpm for 5 min at room temperature, discard the supernatant, and retain the sperm pellet. Repeat this step 3 times.
4. Add 0.1–0.2 mL working wash solution to the EP tube from Step 3 to prepare a sperm suspension.
5. Apply 15 μL of the sperm suspension evenly onto an adhesive microscope slide and air-dry.
6. Add 2–3 drops of fixative solution onto the smear, fix at room temperature for 5–15 min, rinse with distilled water for 5–10 min, and decant excess water.
7. Add 2–4 drops of aniline blue staining solution, stain at room temperature for 5 min, rinse with distilled water for 5 min, and decant excess water.
8. Insert the slide vertically into a container with working elution solution, fully immersing the smear. Incubate at room temperature for 2–5 min.
9. Rinse with distilled water for 1–5 min and decant excess water.
10. Add 2–4 drops of counterstain, stain at room temperature for 2–5 min, rinse with distilled water for 1–5 min.
11. Dry the slide rapidly and examine microscopically. For long-term storage, dehydrate sequentially in 70%, 80%, 95%, and 100% ethanol for 2 min each, clear in xylene or dewaxing/clearing solution, and mount with neutral balsam.
Staining Results:
Sperm with immature nucleoprotein: violet blue or dark blue
Sperm with mature nucleoprotein: red (light blue or colorless without eosin counterstaining)
Precautions:
1. When rinsing the slides with distilled water, control the flow rate carefully to avoid washing off the sperm on the smear area.
2. When adding the staining solution, ensure it fully covers the smear area and mix evenly by pipetting.
3. After discarding excess water, prevent the smear from drying out.
4. For your safety and health, please wear a lab coat and disposable gloves during operation.
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