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Biotinylated ltl

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Biotinylated LTL is a biotin-labeled lectin derived from Lycopersicon esculentum (tomato). Lectins are a class of proteins that bind to specific carbohydrate structures. Biotinylation allows for the detection and visualization of lectin binding in various applications.

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Lab products found in correlation

Streptavidin biotin blocking kit, by Vector Laboratories (2 mentions) 4 6 diamidino 2 phenylindole (dapi), by Merck Group (2 mentions) Eclipse ti, by Nikon (2 mentions) Biotinylated dba, by Vector Laboratories (2 mentions) Lsm 700, by Zeiss (2 mentions) A1r inverted confocal microscope, by Nikon (1 mentions) B 1325, by Abcam (1 mentions) Donkey serum, by Merck Group (1 mentions) Acetylated α tubulin, by Merck Group (1 mentions) Alexa fluor secondary antibody, by Thermo Fisher Scientific (1 mentions) E cadherin, by Abcam (1 mentions) Anti aqp2, by Abcam (1 mentions) Cas block solution, by Thermo Fisher Scientific (1 mentions) Anti cytokeratin, by Agilent Technologies (1 mentions) Anti e cad, by Abcam (1 mentions) Anti cd13, by Abcam (1 mentions) Omniprep solution, by Zytomed Systems (1 mentions) Anti hla, by Abcam (1 mentions) Alexa fluor 488 conjugated anti rabbit and, by Thermo Fisher Scientific (1 mentions) Dapi fluoromount g, by Southern Biotech (1 mentions)

6 protocols using biotinylated ltl

1

Immunofluorescence Staining of Cell Cultures

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Cell cultures were washed once with PBS and fixed in 4% paraformaldehyde for 15 minutes at room temperature (RT). Fixed cells were washed three times in PBS and incubated in blocking buffer (0.3% Triton X-100 and 5% normal donkey serum) for 1 hour at RT. The cells were then incubated with primary antibody overnight at 4°C or for 2 hours at RT in antibody dilution buffer (0.3% Triton X-100 and 1% BSA in PBS). Cells were then washed three times in PBS and incubated with Alexa Fluor 488-, 555-, or 647-conjugated secondary antibodies (1:500) (Life Technologies) in antibody dilution buffer for 1 hour at RT. For immunostaining with biotinylated LTL (Vector Labs, #B-1325), Streptavidin/Biotin Blocking Kit (Vector Labs, #SP-2002) and Alexa Fluor 488- or 647-conjugated streptavidin (Lafe Technologies) were used according to manufacturer’s instructions. Nuclei were counterstained with DAPI (Sigma, #D8417). A list of primary antibodies are shown in Supplementary Data Table 2. Immunofluorescence was visualized using an inverted fluorescence microscope (Nikon Eclipse Ti). Quantification was performed using ImageJ by counting three to five representative fields per experiment at 20X magnification. The sample number of biological replicates in each experiment is shown in figure legends.
Morizane R., Lam A.Q., Freedman B.S., Kishi S., Valerius M.T, & Bonventre J.V. (2015). Nephron organoids derived from human pluripotent stem cells model kidney development and injury. Nature biotechnology, 33(11), 1193-1200.
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2

Immunostaining and Confocal Imaging of Cysts and Organoids

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Immunostaining followed by confocal microscopy was used to localize various proteins and transporters in the cysts and organoids. Prior to staining, an equal volume of 8% paraformaldehyde was added to the culture media (4% final concentration) for 15 mins at room temperature. After fixing, samples were washed in PBS, blocked in 5% donkey serum (Millipore)/0.3% Triton-X-100/PBS, incubated overnight in 1% bovine serum albumin/0.3% Triton-X-100/10μM CaCl2/PBS with primary antibodies, washed, incubated with Alexa-Fluor secondary antibodies (Invitrogen), washed and imaged. Primary antibodies or labels include acetylated α-tubulin (Sigma T7451, 1:5000), ZO-1 (Invitrogen 61-7300, 1:200), Biotinylated LTL (Vector Labs B-1325, 1:500), E-Cadherin (Abcam ab11512, 1:500), SGLT2 (Abcam ab37296, 1:100), laminin-1 (Sigma L9393, 1:50), alpha smooth muscle actin (Sigma A2547, 1:500), CD31 (BD Biosciences 557355, 1:300). Fluorescence images were captured using a Nikon A1R inverted confocal microscope with objectives ranging from 10X to 60X.
Li S.R., Gulieva R.E., Helms L., Cruz N.M., Vincent T., Fu H., Himmelfarb J, & Freedman B.S. (2022). Glucose absorption drives cystogenesis in a human organoid-on-chip model of polycystic kidney disease. Nature Communications, 13, 7918.
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3

Immunofluorescence Staining of Cell Cultures

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Cell cultures were washed once with PBS and fixed in 4% paraformaldehyde for 15 minutes at room temperature (RT). Fixed cells were washed three times in PBS and incubated in blocking buffer (0.3% Triton X-100 and 5% normal donkey serum) for 1 hour at RT. The cells were then incubated with primary antibody overnight at 4°C or for 2 hours at RT in antibody dilution buffer (0.3% Triton X-100 and 1% BSA in PBS). Cells were then washed three times in PBS and incubated with Alexa Fluor 488-, 555-, or 647-conjugated secondary antibodies (1:500) (Life Technologies) in antibody dilution buffer for 1 hour at RT. For immunostaining with biotinylated LTL (Vector Labs, #B-1325), Streptavidin/Biotin Blocking Kit (Vector Labs, #SP-2002) and Alexa Fluor 488- or 647-conjugated streptavidin (Lafe Technologies) were used according to manufacturer’s instructions. Nuclei were counterstained with DAPI (Sigma, #D8417). A list of primary antibodies are shown in Supplementary Data Table 2. Immunofluorescence was visualized using an inverted fluorescence microscope (Nikon Eclipse Ti). Quantification was performed using ImageJ by counting three to five representative fields per experiment at 20X magnification. The sample number of biological replicates in each experiment is shown in figure legends.
Morizane R., Lam A.Q., Freedman B.S., Kishi S., Valerius M.T, & Bonventre J.V. (2015). Nephron organoids derived from human pluripotent stem cells model kidney development and injury. Nature biotechnology, 33(11), 1193-1200.
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4

Immunofluorescence Staining of Tissue Sections

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Five micrometer paraffin sections were pretreated with OmniPrep solution (pH 9.0) at 95°C for 1 h in accordance with the manufacturer’s protocol (Zytomed Systems). Blocking was done using Cas-Block solution (Invitrogen immunodetection kit; 00-8120) for 1 h followed by 1 h incubation with two of the following primary antibodies: anti-OCT4 (Ms [Mouse]; Santa Cruz), anti-HLA (Rb [Rabbit] abcam) anti-cytokeratin (Dako, Glostrup Denmark), anti-LRP (Rb; Novus), biotinylated LTL (Vector Laboratories, Burlingame, CA), biotinylated DBA (Vector Laboratories, Burlingame, CA), anti-EMA (Ms; 1/2 of the prediluted antibody; Cell Marque; 247M-98), anti-CD13 (Rb, 1/400; abcam; 108382), anti-CD31 (Rb, 1/100; abcam; 28364), anti-Aqp1QP1 (Rb, 1/1,000; abcam; 168387), anti-AQP2 (Rb; abcam), anti-E-CAD (Rb; abcam), anti-PODXL (Rb; abcam), HNF1B (Rb; MERCK). Detection was done using Alexa Fluor 488-conjugated anti-rabbit and Alexa Fluor 555-conjugated anti-mouse secondary antibodies (Invitrogen) for 60 min. Mounting medium containing DAPI (Dapi Fluoromount-G; SouthernBiotech; 0100-20) was applied. Slides were analyzed using an Olympus BX51 fluorescence microscope and Olympus DP72 camera or a confocal microscope (ZEISS LSM700). Photo analysis was done using ZEN software.
Omer D., Zontag O.C., Gnatek Y., Harari-Steinberg O., Pleniceanu O., Namestnikov M., Cohen A.H., Nissim-Rafinia M., Tam G., Kalisky T., Meshorer E, & Dekel B. (2023). OCT4 induces long-lived dedifferentiated kidney progenitors poised to redifferentiate in 3D kidney spheroids. Molecular Therapy. Methods & Clinical Development, 29, 329-346.
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5

STING Knockdown Using Lentiviral Vectors

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Lentiviral vector pLKO.1 was used for generating shRNAs targeting STING. Target sequences were selected as follows: shSTING#1, 5′-GCATTACAACAACCTGCTACG-3′; shSTING#2, 5′-GCATCAAGGATCGGGTTTACA-3′.
BFA was purchased from Sigma-Aldrich (St Louis, MO, CAT B6542), hrMANF protein was from R&D (Minneapolis, MN, CAT 3748-MN-050), TAM was from Sigma-Aldrich (CAT T5648) and doxycyline food (1500 ppm, irradiated) was from El Mel (St. Charles, MO, CAT 1814935). Biotinylated LTL was from Vector Laboratories (CAT B-1325-2), Biotinylated DBA was from Vector Laboratories (CAT B-1035-5) and CM-H2DCFDA was from Invitrogen (CAT C6827).
Kim Y., Li C., Gu C., Fang Y., Tycksen E., Puri A., Pietka T.A., Sivapackiam J., Kidd K., Park S.J., Johnson B.G., Kmoch S., Duffield J.S., Bleyer A.J., Jackrel M.E., Urano F., Sharma V., Lindahl M, & Chen Y.M. (2023). MANF stimulates autophagy and restores mitochondrial homeostasis to treat autosomal dominant tubulointerstitial kidney disease in mice. Nature Communications, 14, 6493.
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6

Kidney Organoid Immunofluorescence Imaging

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Brightfield images were taken using the Nikon TS-1000 inverted microscope. For the kidney organoid, antibody staining was performed as described previously20 (link). The following antibodies and dilutions were used: mouse anti-E-cadherin (1:300; BD Biosciences); goat anti-GATA-3 (1:300; R&D Systems); sheep anti-nephrin (1:300; R&D Systems); biotinylated LTL (1:300; Vector Laboratories); and rabbit anti-Jagged1 (1:300; Abcam). Confocal imaging was performed using a ZEISS LSM780 scanning confocal microscope, with a ZEISS Plan-Apochromat 25×/0.8-NA (numerical aperture) multi-immersion objective. Confocal stacks were taken at 1.5-μm Z spacing and exported to the Imaris software (Bitplane) for 3D reconstruction and surface rendering. All other image processing was performed in Fiji31 (link). All immunofluorescence analyses were successfully repeated more than three times; representative images are shown.
Phipson B., Er P.X., Combes A.N., Forbes T.A., Howden S.E., Zappia L., Yen H.J., Lawlor K.T., Hale L.J., Sun J., Wolvetang E., Takasato M., Oshlack A, & Little M.H. (2018). Evaluation of variability in human kidney organoids. Nature methods, 16(1), 79-87.
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