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Sp5x system

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The SP5X is a confocal microscope system designed for advanced imaging applications. It features a modular design that allows for customization to meet specific research needs. The system combines high-performance optics, sensitive detectors, and powerful software to enable detailed analysis of samples.

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Lab products found in correlation

Af software suite, by Leica (5 mentions) Lipofectamine 2000, by Thermo Fisher Scientific (2 mentions) Alexa fluor 488 phalloidin, by Thermo Fisher Scientific (1 mentions) Prolong gold antifade reagent with dapi, by Thermo Fisher Scientific (1 mentions) Alexa fluor 647 anti mouse secondary antibody, by Thermo Fisher Scientific (1 mentions) Mouse monoclonal anti flag antibody, by Merck Group (1 mentions) Recombinant epidermal growth factor, by Thermo Fisher Scientific (1 mentions)

Spelling variants of this product

TCS SP5X system (11 citations) Inverted SP5X Confocal Microscope System (9 citations) SP5X white light laser confocal system (6 citations) TCS 264 SP5X system (3 citations) Leica TCS SP5X confocal system (2 citations)

10 protocols using sp5x system

1

Visualizing Podocyte Filopodia Dynamics

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Human podocyte cells on glass coverslips were transfected with CDC42 constructs and ITSN1 or ITSN2 constructs or pCAGEN-FLAG-mock using Lipofectamine 2000® in six-well plates and were incubated in RPMI with 10% fetal bovine serum for 8 h and then in serum-free medium for 24 h. Cells were fixed in 4% paraformaldehyde (PFA) for 10 min and containing 0.25% Triton X-100. Cells were stained for 60 min at room temperature (RT) with mouse monoclonal anti-FLAG antibodies (1:500, Sigma-Aldrich, F3165) and rabbit polyclonal anti-Myc antibodies (1:500, santa cruz, sc-789), washed with phosphate-buffered saline (PBS) and incubated for 1 h at RT with Alexa Fluor 647 anti-mouse secondary antibody (1:500, life technologies, A31571), Alexa Fluor 594 anti-rabbit secondary antibody (1:500, life technologies, A21207) and Alexa Fluor 488 Phalloidin (1:40, Invitrogen, A12379). Coverslips were then mounted on slides with ProLong Gold antifade reagent with DAPI (Invitrogen). Confocal imaging was performed using Leica SP5X system with an upright DM6000 microscope and images were processed with the Leica AF software suite. Filopodia were defined as thin, finger-like protrusive structures32 (link). The ratio of cells with filopodia was calculated.
Ashraf S., Kudo H., Rao J., Kikuchi A., Widmeier E., Lawson J.A., Tan W., Hermle T., Warejko J.K., Shril S., Airik M., Jobst-Schwan T., Lovric S., Braun D.A., Gee H.Y., Schapiro D., Majmundar A.J., Sadowski C.E., Pabst W.L., Daga A., van der Ven A.T., Schmidt J.M., Low B.C., Gupta A.B., Tripathi B.K., Wong J., Campbell K., Metcalfe K., Schanze D., Niihori T., Kaito H., Nozu K., Tsukaguchi H., Tanaka R., Hamahira K., Kobayashi Y., Takizawa T., Funayama R., Nakayama K., Aoki Y., Kumagai N., Iijima K., Fehrenbach H., Kari J.A., El Desoky S., Jalalah S., Bogdanovic R., Stajić N., Zappel H., Rakhmetova A., Wassmer S.R., Jungraithmayr T., Strehlau J., Kumar A.S., Bagga A., Soliman N.A., Mane S.M., Kaufman L., Lowy D.R., Jairajpuri M.A., Lifton R.P., Pei Y., Zenker M., Kure S, & Hildebrandt F. (2018). Mutations in six nephrosis genes delineate a pathogenic pathway amenable to treatment. Nature Communications, 9, 1960.
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2

Immunofluorescence Imaging of Kidney Proteins

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Immunofluorescence imaging (IF) was performed on rat kidney sections using a Leica SP5X system with an upright DM6000 compound microscope as previously described by the authors (Chaki et al. 2012 (link)). Images were processed with the Leica AF software suite. The following primary antibodies were used: SRGAP1 (Santa Cruz Biotechnology, Cat# sc-81939), ROBO2 (Santa Cruz Biotechnology, Cat# sc-31607), SIX2 (abcam, Cat# ab68908), WT1 (Santa Cruz Biotechnology, Cat# sc-192).
Hwang D.Y., Kohl S., Fan X., Vivante A., Chan S., Dworschak G.C., Schulz J., van Eerde A.M., Hilger A.C., Gee H.Y., Pennimpede T., Herrmann B.G., van de Hoek G., Renkema K.Y., Schell C., Huber T.B., Reutter H.M., Soliman N.A., Stajic N., Bogdanovic R., Kehinde E.O., Lifton R.P., Tasic V., Lu W, & Hildebrandt F. (2015). Mutations of the SLIT2-ROBO2 pathway genes SLIT2 and SRGAP1 Confer Risk for Congenital Anomalies of the Kidney and Urinary Tract. Human genetics, 134(8), 905-916.
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3

Immunofluorescence Staining of Frozen Tissue

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Frozen tissue sections were permeabilized in 0.25%Triton-X100, blocked in 10% donkey serum for an hour at room temperature, and incubated in primary antibody overnight. The cells were incubated in secondary antibodies for 90 min at room-temperature, followed by 5 min. staining with 1 × DAPI/PBS. Confocal imaging was performed using Leica SP5X system with an upright DM6000 microscope and images were processed with the Leica AF software suite.
Braun D.A., Sadowski C.E., Kohl S., Lovric S., Astrinidis S.A., Pabst W.L., Gee H.Y., Ashraf S., Lawson J.A., Shril S., Airik M., Tan W., Schapiro D., Rao J., Choi W.I., Hermle T., Kemper M.J., Pohl M., Ozaltin F., Konrad M., Bogdanovic R., Büscher R., Helmchen U., Serdaroglu E., Lifton R.P., Antonin W, & Hildebrandt F. (2016). Mutations in nuclear pore genes NUP93, NUP205, and XPO5 cause steroid resistant nephrotic syndrome. Nature genetics, 48(4), 457-465.
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4

Immortalized Podocyte Immunostaining Protocol

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For immunostaining human immortalized podocytes were seeded on coverglasses, and grown at permissive temperature. For overexpression studies human podocytes were transiently transfected using lipofectamine 2000® following the manufacturer's instructions. Experiments were performed 24-48 hrs after transfection. Cells were fixed and permeabilized for 10 min using 4% paraformaldehyde and 0.25%Triton-X100. After blocking, cells were incubated with primary antibody over-night at 4°C. The cells were incubated in secondary antibodies for 90 min at room-temperature, followed by 5 min. staining with 1 × DAPI/PBS. Confocal imaging was performed using Leica SP5X system with an upright DM6000 microscope and images were processed with the Leica AF software suite. Immunofluorescence experiments were repeated at least twice in independent experiments.
Braun D.A., Sadowski C.E., Kohl S., Lovric S., Astrinidis S.A., Pabst W.L., Gee H.Y., Ashraf S., Lawson J.A., Shril S., Airik M., Tan W., Schapiro D., Rao J., Choi W.I., Hermle T., Kemper M.J., Pohl M., Ozaltin F., Konrad M., Bogdanovic R., Büscher R., Helmchen U., Serdaroglu E., Lifton R.P., Antonin W, & Hildebrandt F. (2016). Mutations in nuclear pore genes NUP93, NUP205, and XPO5 cause steroid resistant nephrotic syndrome. Nature genetics, 48(4), 457-465.
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5

Overexpression in Human Podocytes

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For overexpression studies, human podocytes were transiently transfected using Lipofectamine 2000 (Thermo Fisher Scientific) according to the manufacturer’s instructions. Experiments were performed 24 to 48 hours after transfection. Cells were fixed for 15 minutes using 4% paraformaldehyde and permeabilized with 0.5% Triton-X 100. After blocking, sections were incubated overnight at 4°C with primary antibody. The cells were incubated in secondary antibodies for 60 minutes at room temperature followed by mounting in hardening medium with 4′,6-diamidino-2-phenylindole (DAPI). Confocal imaging was performed using the Leica (Wetzlar, Germany) SP5X system with an upright DM6000 microscope, images were processed with the Leica AF software suite.
Klämbt V., Mao Y., Schneider R., Buerger F., Shamseldin H., Onuchic-Whitford A.C., Deutsch K., Kitzler T.M., Nakayama M., Majmundar A.J., Mann N., Hugo H., Widmeier E., Tan W., Rehm H.L., Mane S., Lifton R.P., Alkuraya F.S., Shril S, & Hildebrandt F. (2020). Generation of Monogenic Candidate Genes for Human Nephrotic Syndrome Using 3 Independent Approaches. Kidney International Reports, 6(2), 460-471.
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6

Immortalized Podocyte Immunostaining Protocol

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For immunostaining human immortalized podocytes were seeded on coverglasses, and grown at permissive temperature. For overexpression studies human podocytes were transiently transfected using lipofectamine 2000® following the manufacturer's instructions. Experiments were performed 24-48 hrs after transfection. Cells were fixed and permeabilized for 10 min using 4% paraformaldehyde and 0.25%Triton-X100. After blocking, cells were incubated with primary antibody over-night at 4°C. The cells were incubated in secondary antibodies for 90 min at room-temperature, followed by 5 min. staining with 1 × DAPI/PBS. Confocal imaging was performed using Leica SP5X system with an upright DM6000 microscope and images were processed with the Leica AF software suite. Immunofluorescence experiments were repeated at least twice in independent experiments.
Braun D.A., Sadowski C.E., Kohl S., Lovric S., Astrinidis S.A., Pabst W.L., Gee H.Y., Ashraf S., Lawson J.A., Shril S., Airik M., Tan W., Schapiro D., Rao J., Choi W.I., Hermle T., Kemper M.J., Pohl M., Ozaltin F., Konrad M., Bogdanovic R., Büscher R., Helmchen U., Serdaroglu E., Lifton R.P., Antonin W, & Hildebrandt F. (2016). Mutations in nuclear pore genes NUP93, NUP205, and XPO5 cause steroid resistant nephrotic syndrome. Nature genetics, 48(4), 457-465.
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7

Immunofluorescence Staining of Frozen Tissue

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Frozen tissue sections were permeabilized in 0.25%Triton-X100, blocked in 10% donkey serum for an hour at room temperature, and incubated in primary antibody overnight. The cells were incubated in secondary antibodies for 90 min at room-temperature, followed by 5 min. staining with 1 × DAPI/PBS. Confocal imaging was performed using Leica SP5X system with an upright DM6000 microscope and images were processed with the Leica AF software suite.
Braun D.A., Sadowski C.E., Kohl S., Lovric S., Astrinidis S.A., Pabst W.L., Gee H.Y., Ashraf S., Lawson J.A., Shril S., Airik M., Tan W., Schapiro D., Rao J., Choi W.I., Hermle T., Kemper M.J., Pohl M., Ozaltin F., Konrad M., Bogdanovic R., Büscher R., Helmchen U., Serdaroglu E., Lifton R.P., Antonin W, & Hildebrandt F. (2016). Mutations in nuclear pore genes NUP93, NUP205, and XPO5 cause steroid resistant nephrotic syndrome. Nature genetics, 48(4), 457-465.
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8

Podocyte Immunofluorescence Staining Protocol

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Immunostaining was performed as described previously in immortalized human podocytes cell lines. Overexpression experiments were performed 24–48 hrs after transfection with Lipofectamine2000®. Immunostaining followed a standard protocol using permeabilization with 0.1% SDS for cells and 0.025 %Triton-X100 for paraffin embedded tissue sections (rat kidney, day 1 postpartum). Confocal imaging was performed using Leica SP5X system with an upright DM6000 microscope and images were processed with the Leica AF software suite. For lamellipodia staining (Figure 4) cells were grown at 37°C (non-permissive temperature) for 7 days, and were stimulated with recombinant Epidermal Growth Factor (EGF, Thermo Fisher) at a concentration of 100 ng/ml 20 min before staining. Immunofluorescence experiments were performed at least twice independently. Representative images are shown.
Braun D.A., Rao J., Mollet G., Schapiro D., Daugeron M.C., Tan W., Gribouval O., Boyer O., Revy P., Jobst-Schwan T., Schmidt J.M., Lawson J.A., Schanze D., Ashraf S., Boddaert N., Collinet B., Martin G., Liger D., Lovric S., Furlano M., Guerrera I.C., Sanchez-Ferras O., Menten B., Vergult S., De Rocker N., Airik M., Hermle T., Shril S., Widmeier E., Gee H.Y., Choi W.I., Sadowski C.E., Pabst W.L., Warejko J., Daga A., LeBerre T.B., Matejas V., Behnam B., Beeson B., Begtrup A., Bruce M., Ch'ng G.S., Lin S.P., Chang J.H., Chen C.H., Cho M.T., Gipson P.E., Hsu C.H., Kari J.A., Ke Y.Y., Kiraly-Borri C., Lai W.M., Lemyre E., Littlejohn R.O., Masri A., Moghtaderi M., Nakamura K., Praet M., Prasad C., Prytula A., Roeder E., Rump P., Schnur R.E., Shiihara T., Sinha M., Soliman N.A., Soulami K., Sweetser D.A., Tsai W.H., Tsai J.D., Vester U., Viskochil D.H., Vatanavicharn N., Waxler J.L., Wolf M.T., Wong S.N., Poduri A., Truglio G., Mane S., Lifton R.P., Bouchard M., Kannu P., Chitayat D., Magen D., Calleweart B., van Tilbeurgh H., Zenker M., Antignac C, & Hildebrandt F. (2017). Mutations in the evolutionarily highly conserved KEOPS complex genes cause nephrotic syndrome with microcephaly. Nature genetics, 49(10), 1529-1538.
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9

Immunostaining of Frozen Rat Kidney Sections

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For immunostaining of frozen optimal cutting temperature (OCT) compound-embedded tissue sections (adult rat kidney), permeabilization was performed using 0.1% Triton X-100. After blocking, sections were incubated overnight at 4°C with primary antibodies. The cells were incubated in secondary antibodies for 90 min at room temperature, followed by mounting in hardening medium with DAPI. For cell IF, human immortalized podocytes were seeded on coverslips and grown at a permissive temperature. For overexpression studies, human podocytes were transiently transfected using Lipofectamine 2000 (Thermo Fisher Scientific) according to the manufacturer’s instructions. Experiments were performed 24 to 48 hours after transfection. Cells were fixed for 15 min using 4% paraformaldehyde (PFA) and permeabilized as above. Staining was performed similarly to tissue sections. Confocal microscopy imaging was performed using the Leica SP5X system with an upright DM6000 microscope, and images were processed with the Leica AF software suite.
Majmundar A.J., Buerger F., Forbes T.A., Klämbt V., Schneider R., Deutsch K., Kitzler T.M., Howden S.E., Scurr M., Tan K.S., Krzeminski M., Widmeier E., Braun D.A., Lai E., Ullah I., Amar A., Kolb A., Eddy K., Chen C.H., Salmanullah D., Dai R., Nakayama M., Ottlewski I., Kolvenbach C.M., Onuchic-Whitford A.C., Mao Y., Mann N., Nabhan M.M., Rosen S., Forman-Kay J.D., Soliman N.A., Heilos A., Kain R., Aufricht C., Mane S., Lifton R.P., Shril S., Little M.H, & Hildebrandt F. (2021). Recessive NOS1AP variants impair actin remodeling and cause glomerulopathy in humans and mice. Science Advances, 7(1), eabe1386.
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10

Immunofluorescence Imaging of Transfected Podocytes

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For immuno-staining human immortalized podocytes were seeded on coverglasses, and grown at permissive temperature. For overexpression studies human podocytes were transiently transfected using lipofectamine 2000® following the manufacturer’s instructions. Experiments were performed 24–48 h after transfection. Cells were fixed and permeabilized for 10 min using 4% paraformaldehyde and 0.25%Triton-X100. After blocking, cells were incubated over-night at 4 °C. The cells were incubated in secondary antibodies for 90 min at room-temperature, followed by 5 min staining with 1 × DAPI/PBS. Confocal imaging was performed using Leica SP5X system with an upright DM6000 microscope and images were processed with the Leica AF software suite.
Ashraf S., Kudo H., Rao J., Kikuchi A., Widmeier E., Lawson J.A., Tan W., Hermle T., Warejko J.K., Shril S., Airik M., Jobst-Schwan T., Lovric S., Braun D.A., Gee H.Y., Schapiro D., Majmundar A.J., Sadowski C.E., Pabst W.L., Daga A., van der Ven A.T., Schmidt J.M., Low B.C., Gupta A.B., Tripathi B.K., Wong J., Campbell K., Metcalfe K., Schanze D., Niihori T., Kaito H., Nozu K., Tsukaguchi H., Tanaka R., Hamahira K., Kobayashi Y., Takizawa T., Funayama R., Nakayama K., Aoki Y., Kumagai N., Iijima K., Fehrenbach H., Kari J.A., El Desoky S., Jalalah S., Bogdanovic R., Stajić N., Zappel H., Rakhmetova A., Wassmer S.R., Jungraithmayr T., Strehlau J., Kumar A.S., Bagga A., Soliman N.A., Mane S.M., Kaufman L., Lowy D.R., Jairajpuri M.A., Lifton R.P., Pei Y., Zenker M., Kure S, & Hildebrandt F. (2018). Mutations in six nephrosis genes delineate a pathogenic pathway amenable to treatment. Nature Communications, 9, 1960.
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