Staining was performed as previously described (Mitsiadis et al., 2017 (link); Jimenez-Rojo et al., 2019 (link)). Primary antibodies used: polyclonal rabbit anti-Keratin14 (dilution 1:100; Poly19053, Biolegend), polyclonal Goat anti-E-Cadherin (dilution 1:200, AF748, R&D Systems), rat anti-BrdU (dilution 1:50; OBT0300, AbD Serotec), polyclonal Rabbit anti-GFP (dilution 1:100; A11122; Invitrogen). The antibodies were incubated at 4°C overnight. Secondary antibodies used: Goat anti-Rabbit IgG 488, Alexa Fluor Plus 488 (dilution 1:1000; A32731, Invitrogen), Donkey anti-Goat IgG 568, Alexa Fluor Plus 568 (dilution 1:1000; Invitrogen); Goat anti-Rat 546, Alexa Fluor Plus 546 (dilution 1:1000; Invitrogen), Donkey anti-Rabbit IgG 488, Alexa Fluor 488 (dilution 1:1000; A21206: Invitrogen). These antibodies were incubated for 1 h at room temperature (RT).
Af748
Manufactured by R&D Systems
Sourced in United States
AF748 is a laboratory reagent produced by R&D Systems. It functions as a recombinant human protein.
Automatically generated - may contain errors
Lab products found in correlation
A11122, by Thermo Fisher Scientific (1 mentions)
A21206, by Thermo Fisher Scientific (1 mentions)
Poly19053, by BioLegend (1 mentions)
A32731, by Thermo Fisher Scientific (1 mentions)
Ab98952, by BD (1 mentions)
Cytaion5, by Agilent Technologies (1 mentions)
Hoechst 33258, by Thermo Fisher Scientific (1 mentions)
Tcs sp5, by Leica (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (1 mentions)
Ab49602, by Thermo Fisher Scientific (1 mentions)
A21208, by Thermo Fisher Scientific (1 mentions)
A21209, by Thermo Fisher Scientific (1 mentions)
Ma5 14520, by Thermo Fisher Scientific (1 mentions)
A21207, by Thermo Fisher Scientific (1 mentions)
A32795, by Thermo Fisher Scientific (1 mentions)
A32744, by Thermo Fisher Scientific (1 mentions)
A32766, by Thermo Fisher Scientific (1 mentions)
A32814, by Thermo Fisher Scientific (1 mentions)
A32754, by Thermo Fisher Scientific (1 mentions)
Fv1200, by Olympus (1 mentions)
8 protocols using af748
1
Immunostaining of Epithelial Markers
2
Immunofluorescence Staining of Tissues and Cells
3
Immunostaining of Embryonic Gonad Sections
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Embryonic gonads were fixed in 4% PFA for 30 min at 4°C. Gonads were next submerged in 10 and 20% sucrose in PBS for 1 h each at 4°C, and in 30% sucrose in PBS overnight at 4°C. The gonads were then embedded in Tissue-Tek O.C.T. compound (Sakura Finetek) and frozen in liquid nitrogen. Six-micrometer-thick sections of each gonad were applied to glass slides and autoclaved in TRS (Dako). After pre-incubation with 3% skim milk in PBS-T (PBS with 0.1% Tween20 (Sigma-Aldrich)) at RT for 30 min, the sections were reacted with primary antibodies overnight at 4°C at the following dilutions: 1:200 for mouse anti-phospho-histone H3 (ser10) (1:200, Sigma-Aldrich, 06-570), goat anti-CDH1 (1μg/ml, R&D, AF748), rabbit anti-DNMT3L (1:500, provided by Dr. Yamanaka), rabbit anti-STRA8 (1:200, abcam #ab49602), rabbit anti-Ki67 (1:200, Invitrogen #MA5-14520), rat anti-Ki67 (1:100, Invitrogen AB_10854564) and anti-pS6 (1:200, CST #2211). Secondary antibodies labeled with Alexa 488, 594 or 647 (1:1,000, Invitrogen A21207, A21208, A21209, A32766, A32814, A32754, A32744 and A32795) were used. DNA was counter-stained with DAPI (100 ng/ml). Fluorescence microscopy was performed using Olympus FV1200 and images were processed with ImageJ/Fiji (Schindelin et al., 2012 (link)).
4
Immunofluorescence Imaging of Organoids
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For immunofluorescence, organoids were seeded as single cells on a Falcon 4-well Culture slide (FAL354114, In Vitro Technologies, Mt Wellington, Auckland, New Zealand) with 200 cells per well and were induced at day 1 with endoxifen as previously described. If required, organoids were drugged at day 2 for 48 h. At day 4 or 5, organoids were washed for 1 h with PBS and fixed in 1% PFA for 1 h, followed by blocking in 10% FHS/0.5% Triton in PBS. Organoids were then incubated for 3 h, at RT, with primary antibody E-Cadherin 1:100 (AF748, R&D Systems, Minneapolis, MN, USA), Tp53 1:100 (ab246550, Abcam, Cambridge, UK) or Ki-67 1:100 (ab1667, Abcam, Cambridge, UK), followed by a 2 h incubation with the according secondary antibody Donkey anti-Goat Alexa fluor 488 1:400 (A11055, Thermo Fisher Scientific, Waltham, MA, USA) or Goat anti-Rabbit Alexa Fluor 488 1:500 (A11008, Thermo Fisher Scientific, Waltham, MA, USA). Slides were then mounted with ProLong Gold Antifade Mounting with DAPI (P36935, Thermo Fisher Scientific, Waltham, MA, USA) and imaged with a Nikon A1+ inverted confocal laser scanning microscope and a Nikon DSiR2 color 16 MP camera (Otago Micro and Nanoscale Imaging, University of Otago). Organoids were imaged every 10 microns.
5
Proximity Ligation Assay for E-cadherin and β-catenin
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PLA was performed on tissue samples fixed at 4°C for < 24 h in 10% formalin prior to processing using the Duolink Detection kit (Sigma) according to the manufacturer's instructions. Briefly, after citrate buffer-mediated antigen retrieval, the slides were incubated with goat E-cadherin (1/200, R&D Systems AF748) and mouse β-catenin (1/2,000 for mouse tissue, 1/200 for human tissue, #610154, BD Biosciences) overnight. Detection was performed with PLA probes (anti-goat and anti-mouse) conjugated to oligonucleotides. After ligation, amplification detection with a fluorescent probe, slides were imaged on a Zeiss LSM confocal microscope. Z-stacks with 40× objectives were taken. PLA dots in crypts were analysed with ImageJ and either calculated as area fraction or count/nuclei.
6
Immunofluorescence Analysis of Bladder Tissue
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Bladders were aseptically harvested and fixed overnight in methacarn (60% methanol, 30% chloroform, 10% glacial acetic acid), bisected to give two halves per bladder, paraffin-embedded and sectioned. For histopathological examination, slides were stained with hematoxylin and eosin and imaged with a Zeiss Axio Scan Z.1 brightfield slide scanner. Immunofluorescence experiments were conducted as previously described [15 (link)]. Primary antibodies used were uroplakin IIIa (mouse monoclonal, 10R-U103a, Fitzgerald), Trp63 (rabbit polyclonal, GTX102425, GeneTex), E-cadherin (goat polyclonal IgG, AF748, R&D Systems), cytokeratin 5 (chicken polyclonal, 905901, BioLegend) and cytokeratin 20 (mouse monoclonal, M7019, DAKO). Samples were mounted in ProLong Gold Antifade Mountant with DAPI (ThermoFisher Scientific) and fluorescence was visualized on a ZEISS Axioskop Observer.Z1 microscope.
7
Immunostaining Protocol for Tongue and Ganglion Sections
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Immunoreactions were performed as described previously [26, (link)27] (link). Briefly, tongue or ganglion sections were air dried, rehydrated, blocked (in 10% normal donkey serum, 0.3% Triton-X in PBS-X), and incubated overnight at 4˚C with primary antibodies. On the next day, slides were washed and incubated with appropriate secondary antibodies for 1-2 h at room temperature in the dark. Primary antibodies were goat anti-SHH (AF464, 0.1 μg/mL; R&D Systems); rat anti-keratin 8 (TROMA-1, 1:1,000; Developmental Studies Hybridoma Bank); goat anti-Ecadherin (AF748, 1:5,000; R&D Systems) and rabbit anti-RFP (600-401-379, 1:1,000; Rockland). For SHH in tongue sections, the heat-induced antigen-retrieval method [26] (link) was used.
8
Lung Tissue Immunohistochemistry and Immunofluorescence
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Lungs were excised and either embedded in OCT, as previously described, or fixed in 10% (v/v) formalin and embedded in paraffin. Prior to staining, slides with formalin-fixed, paraffin-embedded sections were subject to deparaffinization and antigen retrieval. Prior to staining, slides with fresh-frozen sections were air dried, fixed in ice-cold acetone for 10 min, air dried, and re-hydrated in PBS. Sections were stained with IgG isotype controls (ThermoFisher) and primary antibodies against vimentin (ab92547, Abcam, 1.0 μg/mL), E-cadherin (AF748, R&D Systems, 4.0 μg/mL), α-SMA (ab124964, Abcam, 1.5 μg/mL), CD31 (AF3628, R&D Systems, 10 μg/mL), VE-cadherin (36-1900, Invitrogen, 10 μg/mL), PDGFRβ (3169, Cell Signaling, 1:100), and desmin (ab227651, Abcam, 1.32 μg/mL), as appropriate. For immunohistochemistry with α-SMA, slides were incubated with Rabbit-on-Rodent HRP-Polymer (RMR622, Biocare Medical) at native concentration for 30 min. For immunofluorescence, slides were washed in PBS, incubated with Hoechst (Invitrogen, 5 μg/mL) and the appropriate secondary antibody (Invitrogen, 1:500) for 30 min at room temperature, and washed in PBS. Slides were scanned as previously described.
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