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Fl 1321

Manufactured by Vector Laboratories
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The FL-1321 is a laboratory equipment product manufactured by Vector Laboratories. It serves as a core component for various scientific applications. The detailed specifications and intended use of this product are not available for an unbiased and factual description.

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Lab products found in correlation

Phosphate buffered saline (pbs), by Thermo Fisher Scientific (6 mentions) Donkey serum, by Merck Group (4 mentions) Alexa fluor secondary antibody, by Thermo Fisher Scientific (4 mentions) Bovine serum albumin (bsa), by Merck Group (3 mentions) Lsm 710 confocal microscope, by Zeiss (3 mentions) Hepes buffer, by Thermo Fisher Scientific (2 mentions) Geltrex, by Thermo Fisher Scientific (2 mentions) Geltrex, by BD (2 mentions) Accutase, by Thermo Fisher Scientific (2 mentions) Facsaria cell sorter, by BD (2 mentions) Ab11512, by Abcam (2 mentions) B 1035, by Vector Laboratories (2 mentions) Ab125219, by Abcam (2 mentions) Alexa fluor 488, by Thermo Fisher Scientific (2 mentions) Paraformaldehyde (pfa), by Electron Microscopy Sciences (2 mentions) Lsm 700, by Zeiss (2 mentions) Diva decloaker solution, by Biocare Medical (1 mentions) Lsm710 system, by Zeiss (1 mentions) To pro 3 iodide, by Thermo Fisher Scientific (1 mentions) Axiocam hrc, by Zeiss (1 mentions)

20 protocols using fl 1321

1

Immunofluorescence Analysis of Renal Tissue

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FFPE mouse renal (4 μm) sections were deparaffinized using xylene followed by graded ethanols for rehydration according to the standard protocol. Antigen retrieval was performed using Diva Decloaker solution (Biocare Medical, Concord, CA). After being blocked with blocking buffer containing 5% BSA in PBS-T, the sections were incubated over night, 4 °C with Lotus tetragonolobus agglutinin (LTA) (FL-1321, Vector laboratories, Burlingame, CA) and CaIX (AF2344, R&D Systems, Minneapolis, MN). After washing, secondary antibody donkey -anti rabbit AlexaFluor-594 (A-11056, Life technologies, Carlsbad, CA) and nuclear stain To-pro-3 iodide (Life technologies, Carlsbad, CA) were applied according to the manufacturer’s instructions followed by rinsing and mounting. Sections were subsequently analyzed by confocal scanning using the Zeiss LSM 710 system (Carl Zeiss AG, Oberkochen, Germany).
Johansson E., Rönö B., Johansson M., Lindgren D., Möller C., Axelson H, & Smith E.M. (2016). Simultaneous targeted activation of Notch1 and Vhl-disruption in the kidney proximal epithelial tubular cells in mice. Scientific Reports, 6, 30739.
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2

Kidney Cell Monolayer Preparation

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To prepare monolayers of kidney cells for analysis, freshly isolated organoids were immediately plated onto tissue culture dishes pre-coated with 1 % GelTrex (Thermo Scientific) and cultured for one week in RB, and the resulting epithelial outgrowths were processed for immunoblot and immunofluorescence. Alternatively, to isolate KTECs using flow cytometry, entire organoid cultures were incubated with fluorescein-conjugated LTL (FL-1321, Vector Labs) diluted 1:500 into RB for four hours, dissociated with Accutase, pelleted, resuspended in flow sorting buffer: 1 % FBS, 10 mM HEPES buffer in PBS (Thermo Scientific). LTL+ cells were isolated on a FACSAria Cell Sorter (BD Biosciences) and replated onto GelTrex-coated tissue plates in RB.
Cruz N.M., Song X., Czerniecki S.M., Gulieva R.E., Churchill A.J., Kim Y.K., Winston K., Tran L.M., Diaz M.A., Fu H., Finn L.S., Pei Y., Himmelfarb J, & Freedman B.S. (2017). Organoid cystogenesis reveals a critical role of microenvironment in human polycystic kidney disease. Nature materials, 16(11), 1112-1119.
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3

Immunofluorescent Staining of Kidney Sections

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OCT-embedded kidney sections (4 μm) were incubated with phosphate-buffered saline (PBS) containing 5% horse serum for 1 h at room temperature to block non-specific binding sites. Then the specimens were incubated with primary antibody anti-FABP4 (1:200, 12802-1-AP, Proteintech Group, Chicago, USA) in a humidified chamber overnight at 4°C. After washing, the secondary antibody (1:500, 111-025-003, Jackson ImmunoResearch, West Grove, PA, USA) was used for 1 h. Fluorescein-labeled Lotus tetragonolobus lectin (LTL) (1:400, FL-1321, Vector Laboratories, CA, USA) was applied for identifying proximal tubules. The samples were washed again and then stained with DAPI (1:500, D8200, Solarbio, Beijing, China) and finally sealed with coverslips. Images were acquired by an AxioCamHRc digital camera (Carl Zeiss, Jena, Germany) at ×200 magnification with ZEN 2012 microscopy software (blue edition).
Wang B., Xu J., Ren Q., Cheng L., Guo F., Liang Y., Yang L., Tan Z., Fu P, & Ma L. (2022). Fatty acid-binding protein 4 is a therapeutic target for septic acute kidney injury by regulating inflammatory response and cell apoptosis. Cell Death & Disease, 13(4), 333.
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4

Immunofluorescence Staining of Organoids

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Organoids were fixed in 4% paraformaldehyde for 15 minutes at room temperature. After fixing, samples were washed in PBS, blocked in 5% donkey serum (Millipore)/0.3% Triton X-100/PBS for 1 hour at room temperature, incubated overnight in 3% BSA (Millipore)/PBS with primary antibodies, washed, incubated with Alexa-Fluor secondary antibodies (Invitrogen) and 4′,6-diamidino-2-phenylindole, and washed into PBS for storage. Primary antibodies included PODXL (AF1658, 1:500; R&D) and ECAD (ab11512, 1:500; Abcam). Stains included fluorescein-labeled LTL (FL-1321, 1:500; Vector Labs). Fluorescence images were captured using an inverted Nikon epifluorescence Eclipse Ti or A1R confocal microscope.
Liu E., Radmanesh B., Chung B.H., Donnan M.D., Yi D., Dadi A., Smith K.D., Himmelfarb J., Li M., Freedman B.S, & Lin J. (2020). Profiling APOL1 Nephropathy Risk Variants in Genome-Edited Kidney Organoids with Single-Cell Transcriptomics. Kidney360, 1(3), 203-215.
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5

Immunofluorescence Staining of Organoid and Tissue Sections

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For immunofluorescence, fixed organoid cultures or tissue sections were blocked in 5% donkey serum (Millipore) + 0.3% Triton-X-100/PBS, incubated overnight in 3% bovine serum albumin (Sigma) + PBS with primary antibodies, washed, incubated with Alexa-Fluor secondary antibodies (Invitrogen), washed, and stained with DAPI or mounted in Vectashield H-1000. Labels included fluorescein-labeled LTL (Vector Labs FL-1321, 1:500 dilution) and primary antibodies targeting ZO-1 (Invitrogen 339100, 1:100), hPODXL (R&D AF1658, 1:500), SYNPO (Santa Cruz sc-21537, 1:100), NPHS1 (R&D AF4269, 1:500), NPHS2 (Abcam ab50339, 1:300), PAX2 (Abnova H00005076-M01, 1:100), PAX8 (Proteintech 10336-1-AP, 1:300), and WT1 (Santa Cruz sc-192, 1:100). For PAX2, PAX8, and WT1, paraffin sections were used to determine localization patterns in vivo. Some cytoplasmic staining of these transcription factors was also observed, which was likely an artifact. Antibodies for PAX2 and PAX8 may show some cross-reactivity. Images are representative of stainings from three or more separate experiments.
Kim Y.K., Refaeli I., Brooks C.R., Jing P., Gulieva R.E., Hughes M.R., Cruz N.M., Liu Y., Churchill A.J., Wang Y., Fu H., Pippin J.W., Lin L.Y., Shankland S.J., Vogl A.W., McNagny K.M, & Freedman B.S. (2017). Gene-edited human kidney organoids reveal mechanisms of disease in podocyte development. Stem cells (Dayton, Ohio), 35(12), 2366-2378.
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6

Immunohistochemical Analysis of Key Renal Proteins

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Frozen 8-mm sections were washed with PBS, blocked with 5% (w/v) BSA/PBS (1 h at RT), incubated with a primary antibody anti-Xbp1s (1:50; 619502, BioLegend, San Diego, CA), anti- NKCC2 (1:200; NKCC21-A, Alpha Diagnostic International, San Antonio, TX), or anti-NCC (1:200; SPC-402D, StressMarq, Biosciences Inc., Victoria, Canada) at 4°C overnight, and detected by secondary antibodies conjugated with Alexa Fluor 594 or Alexa Fluor 488 (1:500; Molecular Probes, Eugene, OR). Sections stained with Lotus Tetragonolobus Lectin (LTA) were incubated with fluorescein-labeled LTA (1:500; FL-1321, Vector Laboratories, Burlingame, CA) at RT for 30 min before washing. Sections stained with Dolichos Biflorus Agglutinin (DBA) were incubated with biotinylated DBA (1:400; B-1035, Vector Laboratories, Burlingame, CA) at 4°C overnight, and detected by fluorescein Avidin D (1:500; A-2001, Vector Laboratories, Burlingame, CA).
Ferrè S., Deng Y., Huen S.C., Lu C.Y., Scherer P.E., Igarashi P, & Moe O.W. (2019). Renal tubular cell spliced X-box binding protein-1 (Xbp1s) has a unique role in sepsis-induced acute kidney injury and inflammation. Kidney international, 96(6), 1359-1373.
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7

Fluorescent Labeling of FABP4 for Kidney Imaging

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Recombinant human FABP4 was labeled with Alexa Fluor 647 reactive dye (AF647, A37573, Thermo Fisher Scientific, MA) according to the manufacturer’s protocol26 (link). Ten minutes after intravenous injection of AF647-FABP4 (15 μg/mouse in 200 μl of saline) or saline alone, kidneys were isolated, longitudinally cut into two equal halves, fixed in Carnoy’s solution (60% ethanol, 30% chloroform and 10% glacial acetic acid) for 6 hours and embedded in paraffin. Immunofluorescence analysis was performed with fluorescein LTL (FL-1321, Vector laboratories, CA) or primary antibodies against THP (sc-19554, Santa Cruz, TX) or CalD (ab82812, abcam, MA). Anti-goat and anti-mouse secondary antibodies conjugated with Alexa Fluor 488 (A-110055 and R37120, respectively, Thermo Fisher Scientific, MA) were used to detect THP and CalD, respectively. Images for immunofluorescent analysis were captured with Biozero-8100 immunofluorescence microscope (Keyence Corporation, Osaka, Japan).
Shrestha S., Sunaga H., Hanaoka H., Yamaguchi A., Kuwahara S., Umbarawan Y., Nakajima K., Machida T., Murakami M., Saito A., Tsushima Y., Kurabayashi M, & Iso T. (2018). Circulating FABP4 is eliminated by the kidney via glomerular filtration followed by megalin-mediated reabsorption. Scientific Reports, 8, 16451.
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8

Isolation of LTL+ Kidney Organoid Cells

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For the isolation of LTL+ cells kidney organoids were stained with fluorescein-conjugated LTL (FL-1321, Vector Laboratories). Then specimens were dissociated to single cells using Accumax (07921, Stem Cell Technologies) for 15min followed by 0.25% (wt/vol) trypsin (25300–054, Life Technologies) for 15min at 37 °C. For LTL+ cells isolation FACSDiva software version 8.0.1 (BD Biosciences) was used in the FACS Aria Fusion instrument (BD Biosciences).
Monteil V., Kwon H., Prado P., Hagelkrüys A., Wimmer R.A., Stahl M., Leopoldi A., Garreta E., Hurtado del Pozo C., Prosper F., Romero J.P., Wirnsberger G., Zhang H., Slutsky A.S., Conder R., Montserrat N., Mirazimi A, & Penninger J.M. (2020). Inhibition of SARS-CoV-2 Infections in Engineered Human Tissues Using Clinical-Grade Soluble Human ACE2. Cell, 181(4), 905-913.e7.
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9

Kidney Organoid Immunofluorescence Staining

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Additional kidney organoids were differentiated using the Morizane or Takasato protocols as described in ref. 13 (link). Organoids and human adult kidney were fixed in 4% paraformaldehyde (Electron Microscopy Services), cryoprotected in 30% sucrose solution overnight and embedded in optimum cutting temperature (OCT) compound (Tissue Tek). Organoids and kidneys were cryosectioned at 6 µm thickness and mounted on Superfrost slides (Thermo Fisher Scientific). Sections were washed with PBS (three times, 5 min each), then blocked with 1% bovine serum albumin (Millipore Sigma), permeabilized with 0.1% Triton X-100 in PBS and then stained with primary antibody specific for mouse anti-AQP1 (1:500, Abcam, #ab168387) and Fluorescein labeled LTL (1:500, Vector Labs, #FL-1321). Secondary antibodies included Cy3-conjugated (Jackson ImmunoResearch). Then, sections were stained with DAPI (4′,6′-diamidino-2-phenylindole) and mounted in Prolong Gold (Life Technologies). Images were obtained by confocal microscopy (Nikon C2 + Eclipse; Nikon, Melville, NY).
Nam S.A., Seo E., Kim J.W., Kim H.W., Kim H.L., Kim K., Kim T.M., Ju J.H., Gomez I.G., Uchimura K., Humphreys B.D., Yang C.W., Lee J.Y., Kim J., Cho D.W., Freedman B.S, & Kim Y.K. (2019). Graft immaturity and safety concerns in transplanted human kidney organoids. Experimental & Molecular Medicine, 51(11), 1-13.
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10

Immunofluorescence Analysis of Kidney Organoids

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For immunofluorescence, organoids were fixed on day 18 unless otherwise noted. For fixation, phosphate-buffered saline (PBS; Thermo Fisher Scientific) + 4% paraformaldehyde (Electron Microscopy Sciences) was added to the medium for 15 minutes, after which the samples were washed three times with PBS. Fixed organoid cultures were blocked in 5% donkey serum (Millipore) + 0.3% Triton‐X‐100/PBS, incubated overnight in 3% bovine serum albumin (SigmaAldrich) + PBS with primary antibodies, washed, incubated with AlexaFluor secondary antibodies (Invitrogen), washed, and stained with DAPI or mounted in Vectashield H‐1000. Images were acquired using a Zeiss LSM 700 confocal microscope (Carl Zeiss) and ZEN 3.1 software.
The following primary antibodies were used: anti-ECAD (1:100, ab11512; Abcam), anti-LTL (1:100, FL‐1321; Vector Labs), and anti-nephrosis 1 (NPHS1) (1:100, AF4269; R&D System).
Park K., Lee J.Y., Lee S.Y., Jeong I., Park S.Y., Kim J.W., Nam S.A., Kim H.W., Kim Y.K, & Lee S. (2022). Deep learning predicts the differentiation of kidney organoids derived from human induced pluripotent stem cells. Kidney Research and Clinical Practice, 42(1), 75-85.
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