Solarbio Sperm Leukocyte Peroxidase Stain Kit(n-Toluidine Method)

Product Information

• Product Code: G3410
• Specification: 20T
• Storage: Store at 2-8°C, with a shelf life of 6 months.

Product Content:

Product Description:

Most semen contains white blood cells, mainly polymorphonuclear leukocytes (PMNs, neutrophils). Sometimes, white blood cells can be distinguished from sperm cells and spermatocytes in semen smears using the Papanicolaou staining method. Distinguishing white blood cells mainly relies on differences in staining, size, and morphology of the nucleus. Polymorphonuclear leukocytes are morphologically similar to multinucleated sperm cells, but polymorphonuclear leukocytes stain light blue while sperm cells stain light red. The size of the nucleus also helps in differentiation: the nucleus of mononuclear leukocytes fluctuates widely in size, ranging from approximately 7μm for lymphocytes to over 15μm for macrophages. The number of white blood cells in semen can also be quantified using other techniques.

The principle of the Peroxidase Stain for Seminal White Blood Cells (orthotoluidine method) is that the main type of white blood cells in semen is peroxidase-positive granulocytes, which can be screened using peroxidase staining. This method is rapid, inexpensive, and practical for the initial screening of granulocytes. Although this technique has the advantage of being relatively easy to operate, it cannot detect the following: ① polymorphonuclear leukocytes that have already been activated and released their granules; ② other white blood cell types that do not contain peroxidase, such as lymphocytes, macrophages, and monocytes. This product can be used to differentiate polymorphonuclear leukocytes from multinucleated sperm cells that do not contain peroxidase.

Operating Steps (for reference only):

1. Prepare the orthotoluidine working solution: Add 1ml of saturated ammonium chloride solution, 1ml of EDTA solution, and 10μl of oxidant to 9ml of substrate solution, mix well, and use within 24 hours of preparation.
2. Thoroughly mix the semen sample, mix 0.1ml of semen with 0.9ml of working solution.
3. Vortex the sperm suspension for 10 seconds and let it stand at room temperature for 20-30 minutes. Alternatively, use a tube rocker for continuous agitation.
4. After 20-30 minutes, mix the sperm suspension again and fill the sample into the counting pools on both sides of the hemocytometer.
5. Place the hemocytometer horizontally in a humid chamber at room temperature for at least 4 minutes to allow cells to settle.
6. Use a 200 or 400-fold phase contrast microscope to observe and count at least 200 peroxidase-positive cells.

Specifications:

• Reagent (A): Saturated ammonium chloride solution 2ml, stored at 2-8°C.
• Reagent (B): EDTA solution 2ml, stored at 2-8°C.
• Reagent (C): Orthotoluidine substrate solution 20ml, store at 2-8°C, avoid light.
• Reagent (D): Oxidant 1ml, stored at 2-8°C.

Staining Results:

Peroxidase-positive cells are stained brown, while peroxidase-negative cells remain unstained.

Precautions:

1. Prepare the orthotoluidine working solution just before use and do not store it for an extended period after preparation.
2. If the ambient temperature in the staining room is low, use an incubator or water bath to complete the staining process.
3. When examining counting pools, continue counting at least 200 peroxidase-positive cells per grid until complete grids are counted; do not stop counting in the middle of a grid.
4. For your safety and health, wear lab coats and disposable gloves during operation.
5. This product is for research purposes only and should not be used for clinical diagnosis or other purposes.