Toluidine blue staining kit

TB is a classic nuclear (cationic) dye used for external metachromatic and orthochromatic staining of chromatin,which overall is negatively charged(Sylven et al.,1954; Erenpreisa et al.,1992). Thus,orthochromatic (blue) staining is the result of monomeric dye forms characteristic for either low dve concentrations or low accessibility of sites on the chromatin. Metachromatic(purple)staining,on the other hand, is the result of polymetric dye forms characteristics of cooperative binding to the DNA phosphate residues. The first situation corresponds to highly packaged chromatin of mature sperm cells with their very low stainability by external dyes (Darzynkiewics et al.,1990) The second situation arises when chromatin proteins are more loosely electro statically bound to the DNA and can dissociated from it easily, and DNA stains(e.g.,acridine orange detects denatured or single-stranded DNA).

[ Packing size ] 20 Tests/Kit.

[intended use] For staining detection of sperm nuclear integrity use only.

[Main component]Toluidine blue, ethanol, phosphate buffer.

[Storage conditions and expiry date] Store at 4-6℃ for 1 year.

[applicable instrument ] conventional microscope.

[sample requirements ] Fresh semen.

[Test Method ]

Preparation of reagent before experiment: 

1) . Reagent A: dilute 1:10 with distill water.

2) .Reagent C(fixing solution): just before touse, mixequalvolume Sol.2.5ml of C1 and 2.5ml

Sol. C2 and to cool at 4 ℃ for 30min at least.

3) .Reagent G(stain):just before to use,dilute 0.5 ml of Buffer solution E with 9.5 ml buffersolution F.

Procedure:

1. Ejaculate is allowed to liquefy at 37 ℃ for 30 min,The liquefied sample 1ml is mixed with 5ml reagent A and centrifuge at 2400rpm for 10 min,re-suspended pellet in re-suspending reagent B and adjust to an approximate concentration,200 x 10" cells/ml。

2. Prepare thinsmear on the defatted slide (first immerse in reagent C1 for 30min,after wash in reagent C2, and then air dry for 30 min.

3. Dried smear is fixed with reagent C(fixed solution )and incubating at 4℃ for 30-60min,and then air dried for 12 h.

4. Immerse the slide for to hydrolyze in reagent D(hydrolyzing solution)incubating at 4℃ for 5min.

5. Rinse the slide 3 times x1min in distilled water.

6. Stained with reagent G (TB-O) for 5min at RT.

7. Rinsed the slide briefly in distill water,and air dry.

8. Under light microscopicevaluation,a total of 200-300 spermatozoa are counted in different areas of each slide using oil immersion with ×1,000 magnification,Sperm cell heads with good chromatin integrity are pale blue (TB-), those of diminished integrity are ark blue or violet/purple and categorize (TB+)abnormal cells normal status of TB+ around 25-45%. 

[Reference Value](TB+) Cells >45%: Sperm chromatin integrity poor.

[Notes]The sperm TB staining kit is an in vitro diagnostic reagent,and the results are only for clinical reference.