The protocol for HABP staining was adapted from a published method (Munjal et al., 2021) (link). Embryos were dechorionated at 57 hpf and fixed in 4% paraformaldehyde at room temperature for 2 h. Embryos were washed in PBS for 3 × 5 min, before permeabilisation with ice-cold acetone at −20°C for 7 min. Embryos were washed in PBS-Tr for 3 × 5 min and blocked in PBS-Tr containing 5% BSA for 60 min at room temperature. Embryos were incubated in blocking solution containing biotinylated Hyaluronan Binding Protein (HABP from EMD Millipore, 1:100 dilution of 0.5 μg/μL stock) at 4°C, overnight. Embryos were washed 3 × 15 min with PBS (detergent was avoided due to low binding affinity of HABP to HA). Embryos were incubated with Streptavidin Alexa Fluor 546 (Invitrogen S11225, 1:500) and counterstained for F-actin with Alexa Fluor 488 Phalloidin (8878, Cell Signalling Technology, 1:100 from a 6.6 µM stock). Embryos were washed for 3 × 15 min with PBS before mounting for imaging.
Streptavidin alexa fluor 546
Manufactured by Thermo Fisher Scientific
Sourced in Canada
Streptavidin-Alexa Fluor 546 is a fluorescent conjugate of the protein streptavidin and the Alexa Fluor 546 dye. Streptavidin binds strongly to the biomolecule biotin, and the Alexa Fluor 546 dye provides a red-orange fluorescent signal when excited at the appropriate wavelength.
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Lab products found in correlation
Alexa fluor 488 goat anti rabbit igg, by Thermo Fisher Scientific (2 mentions)
Rabbit against anti cow gfap, by Agilent Technologies (2 mentions)
Goat anti mouse osmr antibody, by LifeSpan BioSciences (2 mentions)
Vectashield mounting media containing dapi, by Vector Laboratories (2 mentions)
Biotinylated anti goat igg, by Vector Laboratories (2 mentions)
Axio imager 2 microscope, by Zeiss (2 mentions)
Anti cd44 fitc, by Thermo Fisher Scientific (2 mentions)
Balb c, by Taconic Biosciences (2 mentions)
Dapi and anti hif1α fluorescein, by R&D Systems (2 mentions)
1ar ultra fast spectral scanning confocal microscope, by Nikon (2 mentions)
Ncr nu nu, by Taconic Biosciences (2 mentions)
Streptavidin alexa fluor 488, by Thermo Fisher Scientific (2 mentions)
Alexa fluor 488 phalloidin, by Cell Signaling Technology (1 mentions)
Ab3080, by Merck Group (1 mentions)
Mowiol, by Merck Group (1 mentions)
Rabbit anti laminin antibody, by Merck Group (1 mentions)
Cm1850, by Leica (1 mentions)
Triton x solution, by Merck Group (1 mentions)
Dulbecco phosphate buffered saline (dpbs), by Thermo Fisher Scientific (1 mentions)
Dapi mounting media, by Vector Laboratories (1 mentions)
16 protocols using streptavidin alexa fluor 546
1
Embryonic Hyaluronan Visualization Protocol
2
Fluorescent Labeling of A. fumigatus Spores
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Spores of A. fumigatus strain TBK1.1 were coated with AlexaFluor546 as described previously [20 (link),33 (link)]. Briefly, isolated spores were incubated with biotin-XX, SSE (Molecular Probes) in the presence of 0.05 M NaHCO3 at 4°C for 2 hours. Spores were pelleted, washed first with 100 mM Tris-HCl at pH 8.0 to deactivate free-floating biotin and next with PBS, followed by incubation with streptavidin-AlexaFluor546 (Invitrogen). Spore concentration was enumerated and resuspended in PBS at 1.5 X 108/ mL. Labeling was confirmed with fluorescence microscopy prior to injections.
3
Quantifying Proliferative Cells in Xenopus Spinal Cord
4
Neutrophil Detection in Tissue Sections
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WT and Lyz2GFP were sacrificed with CO2 and perfused with PBS. Tissues were fixed in 4% paraformaldehyde (PFA) for 18 h at 4°C and subsequently cryopreserved with a sucrose gradient and frozen in optimal cutting temperature compound (OCT; Tissue Tek) in liquid nitrogen. Tissues were cut in 5-µm sections in a cryostat (CM1850; Leica). Neutrophil detection by immunofluorescence was performed in tissues blocked 1 h with 10% BSA and 2% goat serum. Tissues were then incubated with a rabbit anti-laminin antibody (1:300; Sigma) and biotinylated Ly6G (1:50) for 1 h at room temperature. Tissues were washed three times in PBS and incubated with secondary antibodies anti-rabbit Alexa Fluor 635 (Life Technologies) and streptavidin Alexa Fluor 546 (Invitrogen), both diluted 1:500. Tissues were washed in PBS and nuclei were stained with DAPI (1:1,000) before mounting with Mowiol (Sigma).
5
Identification and Enumeration of Cancer Cells and Cancer-Associated Fibroblasts
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Slides were incubated with 100 μL of DPBS (Gibco) for 25 min. The cells were then permeabilized using 100 μL of 0.25% Triton-X solution (Sigma) for 15 min. Following permeabilization, the cells were immersed in 100 μL of 1% BSA for 1 h to block nonspecific interaction. The cells were stained with 100 μL of CD45-biotin (Clone HI30, 10 μg/mL, Biolegend) for 30 min. After washing, the cells were next stained with 100 μL of Streptavidin-Alexa Fluor 546 (10 μg/mL, Invitrogen), anti-Cytokeratin (Clone CAM5.2, 10 μg/mL, BD) in 0.05% Triton-X for 40 min. To identify CAFs, anti-Cytokeratin was replaced by anti-α-Smooth Muscle Actin (α-SMA, Clone 1A4, eBioscience). After each staining step, the cells were washed using 200 μL of DPBS three times. A drop of DAPI mounting media (Vectashield) was placed onto the cells, and the coverslip was placed and sealed with nail polish. Fluorescent micrographs were taken using CellSense software on an Olympus IX81 motorized inverted microscope. Cancer cells were identified using the following criteria: DAPI+, CD45− and CK+. CAF enumeration utilized the following criteria: DAPI+, CD45− and α-SMA+. To quantify the number of CTC clusters, each group of interacting cells involving more than 3 cells was identified as a CTC cluster [23 (link)].
6
Quantifying Intestinal Epithelial Fucosylation
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Mice were treated intraperitoneally (i.p.) with recombinant murine IL-22 (1 µg, Peprotech) or PBS as a control one day before analysis. Immunofluorescent staining for fucosylation of epithelial cells was performed as described (33) . The mucus layer was removed from small intestines with PBS followed by fixation with 4% paraformaldehyde for 4 h. After washing twice with PBS, whole-mount tissues were stained with biotinylated anti-ulex europaeus agglutinin-1 (UEA-1) (1:200, Vector Laboratories, Burlingame, CA, USA) and anti-wheat germ agglutinin (WGA) conjugated to Alexa Fluor 488 (1:200, Invitrogen) overnight at 4°C. Streptavidin-Alexa Fluor 546 (1:400, Invitrogen) was used as a secondary antibody to detect the biotinylated primary antibody. Samples were then stained with DAPI solution (1:5000, Dojindo Laboratories) for 5 min. All images were obtained with a laser scanning confocal microscope (FV3000; Olympus).
7
Immunohistochemical Analysis of GFAP and OSMR
8
Immunohistochemical Analysis of GFAP and OSMR
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Tissues were labelled with rabbit against anti-cow GFAP (DAKO) antibody at a dilution of 1:500 in 5% horse serum in PBS overnight at 4 °C. Next, sections were washed twice for 10 min each in PBS prior to application of Alexa Fluor 488 goat anti-rabbit IgG (Invitrogen, Grand Island, NY) at a dilution of 1:100 in PBS for 3 h. Following secondary antibody incubation, slides were rinsed twice for 10 min each in PBS. Then, tissue was labelled with a goat anti-mouse OSMR antibody (LifeSpan Biosciences, Seattle, WA) at a dilution of 1:200 in 5% horse serum in PBS overnight at 4 °C. Following incubation, slides were rinsed twice for 10 min each in PBS prior before applying biotinylated anti-goat IgG (Vector Laboratories) at a dilution of 1:10,000 in 5% horse serum in PBS for 2 h. Next, slides were rinsed twice for 10 min each in PBS and incubated in Streptavidin Alexa Fluor 546 (Life Technology) at a dilution of 1:100 in PBS for 1 h. Slides were rinsed in PBS for 10 min and then coverslipped with Vectashield Mounting Media containing DAPI (Vector Laboratories). Finally, slides were sealed with acrylic and stored in the dark in a laboratory refrigerator at 4 °C. Images were acquired using a Zeiss Axio Imager 2 microscope and quantified using ImageJ with standard co-localization quantification techniques and the co-localization plugin established by Bolte et al.[15 (link)]
9
In Vivo Nanoparticle Tumor Targeting
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In total, 5 ×106 A549, MDA-MB-468, or MDA-MB-468/Luc cells (1:1 PBS:Matrigel) were injected subcutaneously into the hindflanks of nude mice (NCR nu/nu, Taconic). Tumors were allowed to form for 2–3 weeks. Nanoparticles (8.3 × 1012 NP·kg–1, 5% glucose) were injected via the tail vein into tumor-bearing nude or immunocompetent mice (BALB/c, Taconic). Tumors were harvested after 48 or 72 h and processed by the Swanson Biotechnology Histology Core Facility. Briefly, tumors were formalin-fixed, paraffin-embedded, sectioned (5 μm), deparaffinized, antigen retrieved, and stained using DAPI, biotinylated hyaluronic acid binding protein (Calbiochem), anti-CD44-FITC (Life Technologies), and streptavidin-Alexa Fluor 546 (Life Technologies) or DAPI and anti-HIF1α-fluorescein (R&D Systems). Slides were mounted and imaged using a Nikon 1AR Ultra-Fast Spectral Scanning Confocal Microscope. Whole-animal imaging was performed using a Xenogen IVIS Imaging System (Caliper) with d -luciferin (150 mg kg–1 ip, PerkinElmer) as a bioluminescent substrate. These experiments were approved by the Massachusetts Institute of Technology Committee on Animal Care (CAC).
10
Biotinylation and Accessibility of FAP20-BCCP
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Biotinylation of the FAP20-BCCP in the dmj1-1::FAP20-BCCP axonemes was confirmed by Western blotting with streptavidin–horseradish peroxidase (Thermo Fisher Scientific, Waltham, MA). Accessibility of the BCCP tag in the intact axoneme was assessed using fluorescence-labeled streptavidin. Demembranated axonemes were loaded to a flow cell and blocked with 1% bovine serum albumin (BSA) in HMDEK buffer. Then the flow cell was perfused with streptavidin–Alexa Fluor 546 (Life Technologies, Carlsbad, CA) diluted 1:1000 in the same buffer and washed with the buffer alone. Samples were observed by an IX70 fluorescence microscope as described.
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