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Af software suite

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The AF software suite by Leica is a comprehensive set of tools designed to streamline the autofocus process. The core function of this product is to provide users with advanced autofocus capabilities, ensuring accurate and reliable focus performance across a wide range of applications.

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Lab products found in correlation

Sp5x system, by Leica (5 mentions) 4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (4 mentions) Sp8 system, by Leica (2 mentions) Mouse monoclonal anti flag antibody, by Merck Group (1 mentions) Alexa fluor 647 anti mouse secondary antibody, by Thermo Fisher Scientific (1 mentions) Alexa fluor 488 phalloidin, by Thermo Fisher Scientific (1 mentions) Prolong gold antifade reagent with dapi, by Thermo Fisher Scientific (1 mentions) Recombinant epidermal growth factor, by Thermo Fisher Scientific (1 mentions) Anti acetyl α tubulin, by Cell Signaling Technology (1 mentions) Dab kit, by Vector Laboratories (1 mentions) Anti ehhadh, by Santa Cruz Biotechnology (1 mentions) Anti pankcr, by PTM Biolabs (1 mentions) Anti ck7, by Abcam (1 mentions) Anti sox9, by Santa Cruz Biotechnology (1 mentions)

Close spelling variants of this product

LAS AF software suite Leica Application Suite for Advanced Fluorescence (LAS AF; version 2.35) software Application Suite AF software Leica application suite AF software, version 2.7 Leica Application Suite (LAS) Advanced Fluorescence Lite software (LAS AF Lite, Leica Application Suite (LAS) AF software Application Suite Advanced Fluorescence (LAS AF) software Leica Application Suite AF Lite software Leica Application Suite (LAS AF) microscope software Software Suite

7 protocols using af software suite

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

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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5

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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6

Immunostaining of Nasal Tissues

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Nasal tissues of healthy individuals and the patient carrying TTC12 mutations were seeded on glass cover slips and grown in the presence of 10% FBS under identical culture conditions as previously described [46 (link)]. Then, the tissues were fixed and permeabilized for 10 min using 4% PFA and 0.8% Triton X-100. After blocking (10% normal goat serum [NGS]), slides were incubated with the following primary antibodies: anti-TTC12 (Santa Cruz Biotechnology, sc-390229, 1:100 dilution) and anti-acetyl-α-tubulin (Cell Signaling Technology, #5335, 1:500 dilution). The slides were then incubated with the secondary antibody (Goat anti-rabbit or -mouse IgG, Alexa Fluor 488 or 594; Thermo Fisher Scientific, 1:1000 dilution) for 2 h, followed by staining with DAPI (Thermo Fisher Scientific)/PBS for 6 min. Confocal imaging was performed using an SP8 system (Leica), and images were processed using the Leica AF software suite.
Chen W., Wang F., Zeng W., Zhang X., Shen L., Zhang Y, & Zhou X. (2022). Biallelic mutations of TTC12 and TTC21B were identified in Chinese patients with multisystem ciliopathy syndromes. Human Genomics, 16, 48.
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7

Multiparametric Immunolabeling and Imaging Protocol

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For paraffin sections, dewaxing, hydration, and antigen retrieval steps were performed by following standard protocol. After blocking (10% normal goat serum [NGS]), slides were incubated with the primary antibodies as following: anti-pan-Kcr (PTM Biolabs 501 and 502, 1:1000 dilution); anti-Ehhadh (Santa Cruze sc-393123, 1:200 dilution); anti-CK7(Abcam ab181598, 1:2000 dilution); anti-Sox9 (Santa Cruze sc-166505, 1:100 dilution) overnight at 4 °C. The slides were then incubated with the secondary antibody (Goat anti-rabbit or -mouse IgG, Alexa Fluor 488 or 594; Thermo Fisher Scientific, 1:1000 dilution) for 2 h at 25 °C, followed by staining with DAPI (Thermo Fisher Scientific)/PBS for 6 min. Confocal imaging was performed using an SP8 system (Leica), and images were processed using the Leica AF software suite. For immunohistochemistry, anti-CD3 (99940, 1:150 dilution), anti-CD20 (70168, 1:500 dilution) and anti-CD68 (97778, 1:200 dilution) antibodies are from CST and anti-CD138 (Abcam ab128936, 1:500 dilution). Sections were incubated with biotinylated secondary antibodies and horseradish peroxidase avidin D (HRP, Vector). After three 5-min washes of PBS, the sections were developed with DAB kit (Vector) as standard procedures.
Zhang Y., Chen Y., Zhang Z., Tao X., Xu S., Zhang X., Zurashvili T., Lu Z., Bayascas J.R., Jin L., Zhao J, & Zhou X. (2022). Acox2 is a regulator of lysine crotonylation that mediates hepatic metabolic homeostasis in mice. Cell Death & Disease, 13(3), 279.
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