Repopulated jejunal LNs were fixed 2 hours in 4% PFA, and embedded in Optimal Cutting Temperature (OCT) following overnight infiltration with 30% sucrose. Sections were stained with antibodies against GFAPδ (Bioss, bs-11016R), MAP2 (Abcam, ab32454), GFP (Abcam, ab6556), Keratin-8 (DSHB, TROMA-1), Keratin-5 (Covance, PRB-160P), CD31 (BD, 550274), CD105 (BD, 550546), ER-TR7 (Abcam, ab51824), MUC5AC (Abcam, ab79082), β-catenin (CST, 8480), MUC2 (SCT, sc-15334), or chromogranin A (SCT, sc-18232). Whole intact neurospheres were fixed 30 min in 4% PFA, and embedded in OCT for cryostat sectioning. Frozen sections of neurospheres were successively stained using the following antibodies: Nestin (Ebioscience, 14–5843–82), SOX2 (Abcam, ab97959), Ki-67 (Abcam, ab15580). Alexa Fluor 594 (Invitrogen) secondary antibodies were used for all primary antibodies. Nuclei were counterstained using Hoechst. Donor organs were embedded in paraffin and stained with hematoxylin and eosin (H&E) as described elsewhere.
Ab51824
Manufactured by Abcam
Sourced in United Kingdom, United States
Anti-DDB1 antibody (ab51824) is a rabbit polyclonal antibody that recognizes DDB1 (DNA damage-binding protein 1). DDB1 is a component of the CUL4-DDB1 E3 ubiquitin-protein ligase complex, which is involved in the regulation of cell cycle progression and DNA repair.
Automatically generated - may contain errors
Lab products found in correlation
Ab97959, by Abcam (1 mentions)
Ab15580, by Abcam (1 mentions)
Ab32454, by Abcam (1 mentions)
Alexa fluor 594, by Thermo Fisher Scientific (1 mentions)
Troma 1, by Fortrea (1 mentions)
Fluoview 1000 laser scanning confocal microscope, by Olympus (1 mentions)
Anti ki67 antibody, by Cell Signaling Technology (1 mentions)
A5691, by Merck Group (1 mentions)
Inform 2, by PerkinElmer (1 mentions)
Dia 310, by Dianova (1 mentions)
Ab668, by Abcam (1 mentions)
Ab64613, by Abcam (1 mentions)
Ab84632, by Abcam (1 mentions)
Ab9021, by Abcam (1 mentions)
Ix71 microscope, by Olympus (1 mentions)
Ab45939, by Abcam (1 mentions)
Ab27957, by Abcam (1 mentions)
Ab3201, by Merck Group (1 mentions)
Ab28364, by Abcam (1 mentions)
Alexa fluor 555, by Thermo Fisher Scientific (1 mentions)
7 protocols using ab51824
1
Characterization of Repopulated Jejunal Lymph Nodes
2
Multimarker Immunofluorescence Imaging of Adult Brain
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30 μm parasagittal cryosections from adult brain tissue were stained with anti‐Iba1 (1:500, 019‐19741, Wako, Osaka, Japan), CD31 (1:200, 550274, BD Pharmingen, Heidelberg, Germany), and ER‐TR7 (1:1,000, ab51824, Abcam, Cambridge, UK) for 48 hours (dilution 1:500 at 4°C), followed by Alexa Fluor 488‐ and 568‐conjugated secondary antibody (Life technologies, Darmstadt, Germany) staining, which was added at a dilution of 1:500 overnight at 4°C, as described previously (Erny et al, 2015 (link)). Nuclei were counterstained with DAPI. Imaging was performed on an Olympus Fluoview 1000 confocal laser scanning microscope (Olympus, Hamburg, Germany) using a 20 × 0.95 NA objective. Z stacks were done with 1.14‐μm steps in z direction, and 1,024 × 1,024 pixel resolution was recorded and analyzed using IMARIS software (Bitplane, Zurich, Switzerland). At least three cortical cells were reconstructed per mouse.
3
Quantitative Histological Analysis of Xenograft Tumors
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Microvessel density in the xenograft tumour samples was analysed by immunohistochemistry using the anti-CD31 antibody (DIA-310, Dianova). The area of α-SMA-positive CAFs in the xenograft tumour samples was analysed by immunohistochemistry using an anti-α-SMA antibody conjugated with alkaline phosphatase (A5691, Sigma). The area of Ki67-positive cancer cells in the xenograft tumour samples was analysed by immunohistochemistry using an anti-Reticular Fibroblasts and Reticular Fibres (ER-TR7) antibody (ab51824; Abcam). The overall stroma area in the xenograft tumour samples was analysed by immunohistochemistry using an anti-Ki-67 antibody (#12202; Cell Signaling Technology). Immunostained slides were scanned using the Vectra 2 Automated Slide Imaging System (Perkin Elmer), and quantification of the whole tumour was performed using the inForm 2 software (Perkin Elmer). The area of the tumour region was measured by assessing the haematoxylin-stained area.
4
Comprehensive Tissue Histology Protocols
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For H&E, Masson’s trichrome, and FAH stains, tissue samples were fixed in 10% neutral-buffered formalin (Azer Scientific, Morgantown, PA, USA), paraffin embedded, and sectioned (5 μm). Standard stains were performed with established protocols, whereas IHC for Ki-67 was performed using monoclonal mouse anti-Ki-67 M7240 (Dako, Santa Clara, CA, USA), and FAH was performed as previously described.47 (link) Images were quantified in Aperio ImageScope. For all other IHC stains, tissues were fixed in 4% paraformaldehyde for 4 h, infiltrated with 30% sucrose overnight, and embedded in optimal cutting temperature (OCT) compound for sectioning (5–7 μm). Sections were washed with PBS, blocked with 5% bovine serum albumin (BSA) for 30 min, incubated with primary antibody for 1 h and secondary antibody for 30 min, and counterstained with Hoechst. Images were captured with an Olympus IX71 microscope. The following primary antibodies were used: ER-TR7 and glutamine synthetase cytokeratin 7 (ab51824, ab64613, ab9021; Abcam, Cambridge, MA, USA); CD31 (MCA1746GA; Bio-Rad, Hercules, CA, USA); cytokeratin 18 (ab668, Abcam, Cambridge, MA, USA); and 10830-1-AP, Proteintech, Chicago, IL, USA); and cytokeratin 19 (ab84632; Abcam, Cambridge, MA, USA).
5
Immunostaining Protocol for Cell and Tissue Analysis
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Cells (8×103) were seeded on 10×10 mm coverslips and cultured for 12 hours before staining. Primary antibodies included: rat anti-mouse ER-TR7 (ab51824, Abcam); rabbit anti-mouse CD31 (ab28364, Abcam), vimentin (ab45939, Abcam), FSP-1 (ab27957, Abcam) and CD45 (550539, BD Biosciences); and mouse monoclonal antibody α-SMA (ab5694, Abcam). Secondary antibodies were conjugated with Alexa Fluor 555 or Alexa Fluor 488 (Invitrogen) and nuclei counterstained with 4',6-diamidino-2-phenylindole (Sigma-Aldrich). Slides were assessed by confocal microscopy (FV1000) and images analyzed using the FV10-ASW1.7 Viewer software (both Olympus). Paraffin sections of mouse or human tissues were stained with rabbit anti-mouse TTL (SAB1103321, Sigma-Aldrich) or detyrosinated α-tubulin (ab3201, Millipore) using diaminobenzidine histochemistry kits (Invitrogen). Slides were assessed by brightfield microscopy (DP71, Olympus) and images analyzed using the Image-Pro Plus software (Media Cybernetics).
6
Immunofluorescence Analysis of Glioblastoma Xenografts
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After the last in vivo imaging time point (24 h), U87 tumors and organs (liver, kidneys, and spleen) were harvested (n = 3) and embedded in Tissue-Tek® O.C.T. (Sakura), frozen quickly with 2-methylbutane (Sigma-Aldrich, St. Louis, MO), and maintained at −80 °C. Sections were cut using a cryostat (Leica CM1950; Leica Microsystems Inc., Buffalo Grove, IL, USA) at 8 µm, fixed with cold acetone for 5 min, and stored at −20 °C. The tumor microenvironment in glioblastoma xenografts was analyzed by immunofluorescence (IF), staining with DAPI (0.2 μg/mL) and the following antibodies: rabbit anti-vimentin antibody (2.68 μg/mL, ab92547, Abcam), secondary antibody Alexa Fluor 488 chicken anti-rabbit (4 μg/mL, A-21441, Life Invitrogen); Alexa Fluor 488 Rat anti-mouse F4/80 antibody (10 μg/mL, 123,120, Biolegend); Armenian hamster anti-CD31 antibody (6.7µg/mL, MA3105, Invitrogen) and Alexa Fluor 594 goat anti-hamster (2.5 μg/mL, 405,512, Biolegend); rat anti-reticular fibroblast and reticular fiber antibody (4 μg/mL, ab51824, Abcam) and cyanine3 goat anti-rat (2.5 μg/mL, 405,408, Biolegend). Primary antibodies were incubated for 1 h at room temperature in the dark, followed by 1 h incubation with the respective secondary antibodies under the same conditions. Cryo-tissues were coverslipped with Dako and observed using a confocal microscope (Leica TCS SPE).
7
Multiparametric Immunofluorescence Analysis of Murine Tissues
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The following primary antibodies were used for FACS and IF staining with murine tissues: Ter-119 (TER-119, Biolegend), CD45 (30-F11, Biolegend), EpCAM (G8.8, Santa Cruz Biotechnology), Pdpn (8.1.1, Biolegend), CD31 (390, Biolegend), Procr (eBio1560, Invitrogen), CD90 (53–2.1, Biolegend), CD55 (RIKO-3, Biolegend), αSMA (1A4, Sigma Aldrich), Adamdec1 (Origine #TA323936), Pcolce2 (Proteintech #10607-I-AP), C3 (11H9, Abcam), Sox6 (Abcam #ab30455), ER-TR7 (Abcam #ab51824), Collagen I (Abcam #ab34710), Collagen VI (Abcam #ab6588), and Fibronectin (Abcam #ab2413). The following secondary antibodies were used: donkey anti-rabbit PE (Poly4064, Biolegend), Alexa Fluor 488-, 546-, 568-, and 647-conjugated secondary antibodies were obtained for goat anti-rabbit, goat anti-rat, goat anti-mouse, and goat anti-Syrian hamster from Life Technologies, and DyLight 488- and 649-conjugated secondary antibodies for goat anti-Syrian hamster were obtained from Biolegend. NucBlue viability dye (Invitrogen) was spiked into single-cell suspensions before flow analysis or FACS sorting.
The following probes from Advanced Cell Diagnostics were used for FISH in murine tissues: Pi16 (Mm-Pi16-C2), Grem1 (Mm-Grem1-C3), and Agt (Mm-Agt-C1).
The following probes from Advanced Cell Diagnostics were used for FISH in murine tissues: Pi16 (Mm-Pi16-C2), Grem1 (Mm-Grem1-C3), and Agt (Mm-Agt-C1).
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