Protein samples were resolved in SDS-PAGE and transferred to nitrocellulose membranes, blocked (30 min) in TBST buffer (50 mM Tris/HCl pH 7.5, 0.15 M NaCl, 0.1% (v/v) Tween) with 5% (w/v) dry milk, then incubated in TBST buffer with 5% (w/v) dry milk and 0.5-1 μg/ml antibody (2 h, room temperature (RT) or overnight, 4°C). Protein was detected using either horseradish peroxidase-conjugated secondary antibodies and the enhanced chemiluminescence reagent (Amersham Pharmacia Biotech), or fluorescently labelled secondary antibodies (Invitrogen) and the Odyssey infrared imaging system.
Fluorescently labelled secondary antibody
Manufactured by Thermo Fisher Scientific
Sourced in United States, Germany
Fluorescently labelled secondary antibodies are conjugated with fluorescent dyes. They are used to detect and visualize target proteins in various immunoassays and microscopy applications.
Automatically generated - may contain errors
Lab products found in correlation
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (2 mentions)
Enhanced chemiluminescence reagent, by Cytiva (1 mentions)
Odyssey infrared imaging system, by Thermo Fisher Scientific (1 mentions)
2 4 amidinophenyl 6 indolecarbamidine dihydrochloride dapi, by Merck Group (1 mentions)
Fluorescence microscope, by Olympus (1 mentions)
Dm5500 widefield microscope, by Leica (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions)
Propidium iodide, by Merck Group (1 mentions)
Lsrfortessa cell analyser, by BD (1 mentions)
Facs sortware sorter software, by BD (1 mentions)
Mouse fcr blocking reagent, by Miltenyi Biotec (1 mentions)
Tcs sp5, by Leica (1 mentions)
Isotype control, by R&D Systems (1 mentions)
Fluoromount g, by Thermo Fisher Scientific (1 mentions)
Horseradish peroxidase linked secondary antibody, by Jackson ImmunoResearch (1 mentions)
Synaptophysin, by Merck Group (1 mentions)
F3165, by Merck Group (1 mentions)
A5316, by Merck Group (1 mentions)
S5768, by Merck Group (1 mentions)
Drebrin, by Abcam (1 mentions)
19 protocols using fluorescently labelled secondary antibody
1
Western Blot Protein Detection Protocol
2
HMGB-1 Immunofluorescence Staining
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Cells were washed with PBS and fixed with 4% paraformaldehyde (PFA). After incubation with 0.1% Triton X‐100 for 2 minutes on ice, the cells were washed twice with cold PBS. Then, the cells were blocked with bovine serum albumin (BSA) and incubated with HMGB‐1 at 37°C for 30 minutes. After washing with PBS, the cells were incubated with appropriate fluorescently labelled secondary antibodies (Invitrogen) at 30°C in the dark. Finally, 2‐(4‐Amidinophenyl)‐6‐indolecarbamidine dihydrochloride (DAPI, Sigma‐Aldrich) was used to stain the cell nuclear. The cells were visualized under a fluorescence microscope (Olympus).
3
Immunofluorescence Staining of FFPE Tissue
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Formalin-fixed and paraffin-embedded tissue were sectioned, de-paraffinized and antigens were retrieved using boiling 10 mM citrate (pH 6). Subsequently, sections were permeabilized with 0.2% Triton X100/PBS (5 min, RT) and blocked with 10% serum/PBS (1 h, RT). Primary antibody staining was overnight (4 °C) using anti-acetylated α-tubulin (Sigma T6793) and anti-pericentriolar material 1 (PCM1; CST #5213) antibodies, followed by a 1–2 h incubation at RT with fluorescently labelled secondary antibodies (Invitrogen). Sections were embedded in Vectashield/DAPI and visualized on a Leica DM 5500 Wide field microscope (Leica Microsystems, Wetzlar, Germany) using 3D deconvolution. Resulting z-stacks are presented as maximum intensity projections. The use of human patient material through the Marburg Biobank was approved by the local ethics committee at the University Hospital Marburg.
4
Tumor Cell Immunophenotyping and Sorting
5
Immunofluorescence Analysis of Activin-A
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For immunofluorescence experiments, lung sections were deparafinized, rehydrated and blocked in 10% donkey serum, diluted in 0.1% Triton-X. Lung sections were incubated overnight at 4 °C with a primary antibody against mouse/human activin-A or isotype controls (R&D Systems). Lung sections were incubated with fluorescently-labelled secondary antibodies (Invitrogen) for 1 h at room temperature (RT). Nuclear staining and mounting were carried out using DAPI (ThermoFischer Scientific) and Fluoromount-G (eBioscience). Image acquisition was performed using a confocal laser scanning microscope (Leica TCS SP5). Immunofluorescence data analysis was performed with the Image J software.
6
Western Blot Protein Detection
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Protein samples were resolved in sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) and transferred to nitrocellulose membranes, blocked (30 min) in TBST buffer (50 mM Tris/HCl pH 7.5, 0.15 M NaCl, 0.05% (vol/vol) Tween) with 5% (wt/vol) dry milk, then incubated in TBST buffer with 5% (wt/vol) dry milk and 0.5–1 μg/ml antibody (2 hr, room temperature [RT] or overnight, 4°C). Protein was detected using fluorescently labelled secondary antibodies (Invitrogen) and the Licor Odyssey infrared imaging system.
7
Immunodetection of cellular proteins
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Antibodies to CHCHD10 (1:500, 25,671-1-AP, Proteintech, Rosement, IL, USA), M2 (1:1,000, F3165, Sigma-Aldrich), TOM20 (1:1,000, FL-145, Santa Cruz Biotechnology, Dallas, TX, USA), Drebrin (1:500, ab12,350, Abcam, Cambridge, MA, USA), Synaptophysin (1:500, S5768, Sigma-Aldrich), TDP-43 (1:500, MABN45, Merck Millipore, Darmstadt, Germany), LaminB1 (1:1,000, D4Q4Z, Cell Signaling, Danvers, MA, USA), Actin (1:3,000, A5316, Sigma-Aldrich), Actin (1:1,000, sc-1,616, Santa Cruz, Dallas, TX, USA), tRFP (1:1,000, AB233, Evrogen, Moscow, Russia), horseradish peroxidase-linked secondary antibodies (1:5,000, Jackson ImmunoResearch, West Grove, PA, USA), and fluorescently labelled secondary antibodies (1:1,000, Invitrogen) were obtained from the indicated sources.
8
Antibodies Used for Immunofluorescence
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Antibodies used in this study include in-house antibodies against clathrin, AP-5 ζ (monoclonal antibody used for IF), µ5 and SPG11 monoclonal (4 (link)) and spastizin [PER antibody (49 (link))] and commercial antibodies against spastizin (Atlas HPA035693), EEA1 (BD Transduction Labs E41120), LAMP1 (Abcam ab24170 and H4A3), CIMPR (2G11; Calbiochem 444105), AP-2 µ2 (AP50; BD Transduction Labs 611351), LC3 (4E12; MBL M152-3B), LBPA (Jean Gruenberg), CD63 (H5C6; BD Biosciences), tubulin (DM1A, Abcam ab7291) and actin (C4, Abcam ab3280). Horse radish peroxidase-labelled secondary antibodies were purchased from Sigma and fluorescently labelled secondary antibodies from Invitrogen.
9
Affinity Purification of SARS-CoV-2 Nucleocapsid Protein Complexes
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DF‐12K medium, l ‐glutamine, FBS and trypsin were from Sigma‐Aldrich. Antibody against SARS‐CoV‐2 Nucleocapsid Protein (Cat. No. 26369) and USP10 (Cat. No. 5553S) were from Cell Signaling. Antibodies against TIA1 (Cat. No. ab140595), G3BP1 (Cat. No. ab56574), His‐tag (Cat. No. ab18184), YB‐1 (Cat. No. ab76149) and YTHDF3 (Cat. No. ab220161) were from Abcam. Antibodies against G3BP2 (Cat. No. NBP1‐82976), CAPRIN1 (Cat. No. NBP2‐22238) and DDX6 (Cat. No. NB200‐191) were from Novus Biologicals. Antibody against β‐Actin (Cat. No. sc‐47778) was from Santa Cruz Biotechnology. Fluorescently labelled secondary antibodies (mouse, Alexa Fluor 488/594; rabbit, Alexa Fluor 488/594) and Protein A Magnetic Beads were from Life Technologies. 1,6‐Hexanediol was from Sigma. TransIT‐2020 transfection reagent was from Mirus. HisPur™ Ni‐NTA Spin Purification Kit (1 mL), Zeba Spin desalting columns (7K MWCO 2 mL) and LightShift Chemiluminescent RNA EMSA kit were from Thermo Fisher Scientific.
10
Microdevice Immunofluorescence Characterization
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After 2 and 7 days of culture, immunofluorescence analysis was performed directly within the microdevices. The samples were fixed in 4% paraformaldehyde (PFA) for 45 minutes. In order to permeabilize and block non-specific bindings, the samples were incubated for 45 minutes at 4 1C in 5% donkey serum, 2% bovine serum albumin (BSA) and 0.3% Triton (Sigma) in PBS solution. Samples were then incubated overnight at 4 1C with primary antibodies. Cell proliferation was evaluated after 2 days by staining with polyclonal rabbit anti-rat Ki67 (dilution 1 : 100, Abcam). Monoclonal IgG2a anti-mouse alpha-smooth muscle actin (a-SMA, dilution 1 : 400) was used to assess the transition from fibroblast towards myofibroblast phenotype after 7 days of culture. Monoclonal IgG1 anti-mouse collagen type I (dilution 1 : 100, Abcam), polyclonal anti-rabbit aggrecan (dilution 1 : 200, Abcam) and polyclonal anti-rabbit fibronectin (dilution 1 : 200, Abcam) were chosen to define the extracellular matrix (ECM) deposition after 7 days of culture. At every time point, DAPI staining was used to recognise the nuclei. Fluorescently labelled secondary antibodies (Life Technologies) were used at 1 : 200 for 1 hour at room temperature.
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