Alexa fluor 488 conjugated lectin pna

Manufactured by Thermo Fisher Scientific

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Alexa Fluor 488-conjugated lectin PNA is a fluorescently labeled lectin derived from the peanut plant (Arachis hypogaea). It binds specifically to galactose-beta-1,3-N-acetylgalactosamine residues, which are commonly found on the surface of cells.

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Lab products found in correlation

Glycine, by Kanto Chemical (1 mentions) Polyvinyl alcohol (pva), by Fujifilm (1 mentions) Triton x 100, by Fujifilm (1 mentions) Vectashield mounting medium, by Funakoshi (1 mentions) Tcs sp5 confocal laser microscope, by Leica camera (1 mentions) Paraformaldehyde (pfa), by Fujifilm (1 mentions) Bovine serum albumin (bsa), by Merck Group (1 mentions) Polyvinyl alcohol (pva), by Merck Group (1 mentions) Ab15093, by Abcam (1 mentions) 4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (1 mentions) Sc 1237, by Santa Cruz Biotechnology (1 mentions) Hpa001499, by Merck Group (1 mentions) Alexa fluor 488 or 594, by Thermo Fisher Scientific (1 mentions) Fluoromount, by Diagnostic BioSystems (1 mentions) Gentamicin, by Thermo Fisher Scientific (1 mentions) β mercaptoethanol, by Thermo Fisher Scientific (1 mentions) Acrylamide bis solution, by Bio-Rad (1 mentions) Alexa fluor 488 conjugated anti rabbit igg, by Thermo Fisher Scientific (1 mentions) Triton x 100, by Bio-Rad (1 mentions) Peroxidase conjugated anti rabbit igg, by GE Healthcare (1 mentions)

4 protocols using alexa fluor 488 conjugated lectin pna

Cortical Granule Lectin Staining in Oocytes

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Cortical granule (CG) lectin was stained as described previously.22, 23 Oocytes were fixed with 4% paraformaldehyde (Wako Pure Chemical Industries, Ltd, Osaka, Japan) in Dulbecco's phosphate‐buffered saline (PBS) for 1 h at room temperature, followed by incubation in blocking solution, which comprised PBS containing 0.2% polyvinyl alcohol (PVA; Sigma‐Aldrich), 0.3% bovine serum albumin (BSA) (Sigma‐Aldrich), and 100 mM glycine (Kanto Chemical, Tokyo, Japan) for 5 min. Subsequently, oocytes were washed with PBS/PVA containing 0.1% Triton X‐100 (Wako Pure Chemical Industries, Ltd) and incubated with Alexa Fluor 488‐conjugated lectin PNA (1:200; Molecular Probes, OR) for 30 min at room temperature. After washing in PBS/PVA containing 0.01% Triton X‐100 and 0.3% BSA, oocytes were mounted onto glass slides using VECTASHIELD Mounting Medium (Funakoshi, Tokyo, Japan). Images were obtained using a TCS SP5 confocal laser microscope (Leica, Tokyo, Japan).

Shiina K., Komatsu M., Yokoi F., Bai H., Takahashi M, & Kawahara M. (2019). Overgrowth of mice generated from postovulatory‐aged oocyte spindles. FASEB bioAdvances, 1(7), 393-403.

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Immunohistochemical Analysis of Key Proteins

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Using the formalin-fixed paraffin sections, immunohistochemistry (IHC) was performed as described previously (Nakata et al., 2015b (link), 2017b (link)). The sections were incubated with antibodies against ZBTB16 (1:200 dilution; HPA001499, Sigma-Aldrich, St. Louis, MO, United States), GATA4 (1:50; sc-1237, Santa Cruz Biotechnology, Dallas, TX, United States), or SYCP3 (1:400; ab15093, Abcam, Cambridge, United Kingdom) followed by secondary antibodies labeled with Alexa Fluor 488 or 594 (1:400, Molecular Probes, Eugene, OR, United States), or with Alexa Fluor 488-conjugated lectin PNA (1:400, Molecular Probes). All sections were counterstained with DAPI (300 nM; Molecular Probes), mounted on glass slides with Fluoromount (Diagnostic BioSystems, Pleasanton, CA, United States), and examined with a fluorescence microscope (BX51; Olympus, Tokyo, Japan). Fluoromount was then removed and the sections were washed and stained with hematoxylin and eosin (HE).

Nakata H., Nakano T., Iseki S, & Mizokami A. (2020). Three-Dimensional Analysis of Busulfan-Induced Spermatogenesis Disorder in Mice. Frontiers in Cell and Developmental Biology, 8, 609278.

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Sperm Characterization in Trpm7 Mutant Mice

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Spermatozoa of Trpm7^fl/fl and Trpm7-Sp cKO were examined and compared following standard methods.^{80 (link)} Sperm concentration and morphology were evaluated in non-capacitated (NCAP) spermatozoa fixed after swimming out. After washing, they were resuspended in PBS, and 200 were analyzed per sample using contrast-phase microscopy at 400X. Sperm motility was determined in NCAP and in capacitated spermatozoa (CAP) by loading 35 μL into a pre-warmed chamber slide (Leja, 100 μm in depth) at 37 °C. CEROS computer-assisted sperm analysis system, CASA (Hamilton Thorne Research, Beverly, MA), was used with the default setting to analyze at least five microscopy fields. The acrosome status in NCAP and CAP sperm was examined using fixed samples spread on poly-L-lysine treated slides and air-dried. Following permeabilization and washes, sperm were incubated with Alexa Fluor 488-conjugated PNA lectin (Invitrogen) 1:100 in PBS at RT and protected from light for one h. Slides were treated with mounting medium (Vectashield, Vector Laboratories, Newark, CA) and permanently mounted with coverslips sealed with nail polish.

Gupta N., Soriano-Úbeda C., Stein P., Savy V., Papas B.N., Ardestani G., Carvacho I., Alfandari D., Williams C.J, & Fissore R.A. (2023). Essential role of Mg2+ in mouse preimplantation embryo development revealed by TRPM7 chanzyme-deficient gametes. Cell reports, 42(10), 113232.

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Immunofluorescence Analysis of TSSK3 in Cells

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Reagents were obtained from various sources and details are provided below, under the subsections pertaining to each experimental procedure. Bovine serum albumin (A-7906, for blocking non-specific binding), Tween-20 (P-7949), Immobilon-P PVDF membrane (Millipore) and poly-L-lysine (P-8920) were purchased from Sigma-Aldrich (St. Louis, MO). Paraformaldehyde (16% solution, EM Grade), and β-mercaptoethanol were purchased from Fisher Scientific (Waltham, MA). Triton X-100 and 30% acrylamide/Bis solution were obtained from BioRad. Peroxidase-conjugated anti-mouse IgG (Jackson Immunoresearch, West Grove, PA) and peroxidase-conjugated anti-rabbit IgG (GE Healthcare, Pittsburg, PA) were used. AlexaFluor488-conjugated PNA lectin, AlexaFluor488-conjugated anti-rabbit IgG, Hoechst 333452 and gentamicin (10 mg/ml) were purchased from Invitrogen (ThermoFisher Scientific, Grand Island, NY). Anti-TSSK3 rabbit polyclonal antibodies AP7246a and AP17810b were obtained from Abcepta (San Diego, CA). A rabbit polyclonal antibody against alpha/beta-tubulin was obtained from Cell Signaling Technology (Danvers, MA).

Nayyab S., Gervasi M.G., Tourzani D.A., Caraballo D.A., Jha K.N., Teves M.E., Cui W., Georg G.I., Visconti P.E, & Salicioni A.M. (2021). TSSK3, a novel target for male contraception, is required for spermiogenesis.. Molecular reproduction and development, 88(11), 718-730.

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