Acrosome reaction kit, method for inducing acrosome reaction and , and method for evaluating acrosome reaction condition
A technology of acrosome reaction and kit, which is applied in the field of sperm acrosome function screening, can solve the problems of high experimental intensity, poor methodological stability and repeatability, and affect the accuracy of result interpretation, so as to ensure accuracy and repeatability , ease the work intensity, compact test process effect
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Embodiment 1
[0089] 1. Kit preparation
[0090] 1. Select the general product in the prior art for use, prepare 80% density gradient solution, 40% density gradient solution, sperm washing solution A (pH is 7.2~7.6), sperm culture fluid, 5 × sperm washing solution according to the above specifications and dosage Solution B, calcium ionophore, control solution, fixative solution, tissue slide, long-shaped cover slip. The calcium ionophore is the A23187 calcium ionophore provided by Sigma. The materials of these existing products may not be included in the kit, and the users prepare these materials by themselves before the experiment.
[0091] 2. Prepare tissue slice preservation solution. Mix 0.5ml of glutaraldehyde, 5ml of methanol, 2ml of ethanol, 0.2g of PVP (polyvinylpyrrolidone) and 1ml of formalin with purified water to obtain 100ml of tissue slice preservation solution.
[0092] 3. Preparation of fluorescent conjugates. First prepare the fluorescent conjugate preservation solution...
Embodiment 2
[0151] 1. Kit preparation
[0152] The preparation of the kit in Example 2 is roughly the same as in Example 1, except that when preparing the fluorescent conjugate, during the preparation of the fluorescent conjugate preservation solution, fluorescein markers: 5 ng, methacrylic acid, methyl methacrylate Ester copolymer (1:2): 1.5g, Tris buffer: 2.5g, NaCl: 0.9g, casein: 0.3g, sodium salicylate: 1.4mg, preservative Proclin300: 60ul, the preparation method is the same as in Example 1 . When preparing fluorescent mounting solution, take N-(1,3-dimethylbutyl)-N'-phenyl-p-phenylenediamine: 0.2g, polyvinyl alcohol PVA: 0.9g, polyethylene glycol 8000: 0.8 g, Tris buffer solution: 2.5 g, NaCl: 1.0 g, bovine serum albumin BSA provided by Sigma Company: 3 g, thimerosal provided by Sigma Company 0.03 g, glycerol: 30%, and the preparation method is the same as in Example 1.
[0153] 2. Detection of induced sperm acrosome reaction
[0154] (1) Reagent preparation
[0155] With embodim...
Embodiment 3
[0178] 1. Kit preparation
[0179] The preparation of the kit in Example 2 is roughly the same as in Example 1, except that when preparing the fluorescent conjugate, during the preparation of the fluorescent conjugate preservation solution, fluorescein markers: 10 ng, methacrylic acid, methyl methacrylate Ester copolymer (1:2): 2.4g, Tris buffer: 3.5g, NaCl: 1.5g, casein: 0.5g, sodium salicylate: 2.0mg, preservative Proclin300: 100ul, the preparation method is the same as in Example 1 . When preparing fluorescent mounting solution, take N-(1,3-dimethylbutyl) N'-phenyl-p-phenylenediamine: 0.5g, polyvinyl alcohol PVA: 1.6g, polyethylene glycol 8000: 1.5g , Tris buffer: 5.0g, NaCl: 1.6g, bovine serum albumin BSA: 5g, NaN 3 0.05g, glycerol: 60%, and the preparation method is the same as in Example 1.
[0180] 2. Detection of induced sperm acrosome reaction
[0181] (1) Reagent preparation
[0182] With embodiment 1.
[0183] (2) Carry out testing procedures
[0184] Steps 1...
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