Sperm were incubated in a 100-μL drop of cTYH for 0 or 60 min. After incubation, the concentration of each sperm suspension was measured using a PiCOSCOPE® (USHIO, Japan) and equalized. Aliquots of the sperm suspension were collected by centrifugation at 14,000 × g for 5 min. Subsequent to washing with 1 mL of phosphate-buffered saline, the sperm pellet was resuspended in Laemmli sample buffer and 2-mercaptoethanol and boiled for 5 min. After disruption of sperm sample by ultrasonication for 1 min, SDS-PAGE was performed using Mini-PROTEAN® Tetra Vertical Electrophoresis Cell (Bio-Rad, USA), and proteins were transferred to a Trans-Blot® Turbo Mini PVDF Transfer Pack (Bio-Rad). Western blot analysis was performed using the SNAP i.d.® 2.0 Protein Detection System (Merck Millipore, Germany). The protein-transferred membrane was blocked with PO Noise Cancelling Reagents for Phosphoprotein Detection using chemiluminescence or fluorescence techniques (Bløk; Merck Millipore) at room temperature. After removing the blocking solution, the membrane was immunoblotted with a monoclonal antibody against phosphotyrosine (4G10® Platinum, Anti-Phosphotyrosine Antibody; Merck Millipore) for 10 min at room temperature and horseradish peroxidase-conjugated secondary antibodies for 10 min at room temperature. α-Tubulin was used as the internal control.
Picoscope
Manufactured by Ushio
Sourced in Japan
The PiCOSCOPE is a compact and high-performance microscope system designed for laboratory use. It features a built-in camera and delivers precise optical performance for a range of microscopy applications.
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Lab products found in correlation
3 protocols using picoscope
1
Sperm Tyrosine Phosphorylation Assay
2
Platelet Suspension Spectrophotometric Analysis
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P-PRP and other platelet suspensions were serially diluted with equal volume of acellular plasma or the corresponding buffer solutions. Series of diluted platelet suspensions were measured using a pocketable spectrophotometer (PiCOSCOPE, Ushio Inc., Tokyo, Japan) [6 (link)]. The spectrophotometer can be operated by remote control through a specific application installed on smart devices, including the iPad Air (Apple, Cupertino, CA, USA). Platelet suspensions were transferred into 0.2 mL highly transparent PCR tubes (Nippon Genetics Co., Ltd., Tokyo, Japan) and treated with 5 μM ADP (Wako Pure Chemicals, Osaka, Japan).
To prepare dysfunctional platelet models, we pretreated P-PRP with 0.1 mg/mL aspirin (acetylsalicylic acid; Wako Pure Chemicals, Osaka, Japan) or 10 μM H2O2 (Wako) for 30 min at 22–24 °C. The absorbance was measured at an interval of one minute at 615 nm (range of wavelength: 570–660 nm). At the end of measurement, each blank was measured as the absorbance of 100% aggregation.
To prepare dysfunctional platelet models, we pretreated P-PRP with 0.1 mg/mL aspirin (acetylsalicylic acid; Wako Pure Chemicals, Osaka, Japan) or 10 μM H2O2 (Wako) for 30 min at 22–24 °C. The absorbance was measured at an interval of one minute at 615 nm (range of wavelength: 570–660 nm). At the end of measurement, each blank was measured as the absorbance of 100% aggregation.
3
Platelet-Rich Plasma Characterization by SPM
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P-PRP and L-PRP preparations were serially diluted with the corresponding amount of PPP. The series of P-PRP and L-PRP dilutions were first subjected to measurement using the AHA and subsequently subjected to measurement with a compact scanning probe microscope (SPM; PiCOSCOPE, Ushio Inc., Tokyo, Japan) (Fig. 1 ). The SPM can be operated by remote control through a specific application installed on smart devices, including the iPad Air (Apple, Cupertino, CA, USA). PRP samples were transferred into 0.2 mL highly transparent PCR tubes (Nippon Genetics Co., Ltd., Tokyo, Japan) and were measured at 615 nm (range of wavelength 570–660 nm).![]()
A compact SPM with its remote controller installed on an iPad Air. iPhones and other Android devices can be used instead of the iPad Air
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