RAW264.7 macrophages were seeded overnight on glass coverslips in a 24‐well plate. The next day, cells were exposed to the indicated concentrations of CuO NPs or CuCl2 for 6 h. Some cells were preincubated with 4-IPP (50 µM) (Sigma-Aldrich) for 1 h prior to exposing to CuO NPs or CuCl2. After exposure, coverslips were washed twice with 1X PBS and incubated with MitoTracker™ Deep Red (Thermo Fisher Scientific) for 15 min at 37 °C in CO2 incubator. Cells were washed again with 1X PBS and fixed with 4% formaldehyde for 15 min at room temperature. Next, the cells were permeabilized in 0.1% of Triton‐X 100 (Sigma‐Aldrich) for 15 min, followed by blocking with 10% of goat serum (Abcam) supplemented with 0.1% of Triton‐X100 for 1 h. Cells were then incubated overnight at 4 °C with rabbit monoclonal anti-SOD1 antibody (1:200, Abcam) prepared in the antibody buffer (8% of goat serum and 0.1% of Triton‐X‐100). The following day, the cells were rinsed in PBS and incubated with Alexa Fluor® 488 conjugated goat anti‐rabbit antibody (1:500, Life Technologies, Thermo Fisher Scientific) for 1 h. Coverslips were then mounted with Prolong Gold antifade reagent with DAPI (Invitrogen) and imaged using a Zeiss LSM900-Airy confocal laser scanning microscope and ZEN software (Zeiss).
Goat serum
Manufactured by Abcam
Sourced in United Kingdom, United States, China
Goat serum is a biological product derived from the blood of goats. It is commonly used as a supplement in cell culture media to support the growth and proliferation of various cell types in laboratory settings.
Automatically generated - may contain errors
Lab products found in correlation
Triton x 100, by Merck Group (13 mentions)
Goat serum, by Merck Group (11 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (8 mentions)
Goat serum, by Thermo Fisher Scientific (8 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (8 mentions)
Bovine serum albumin (bsa), by Merck Group (5 mentions)
Ab150077, by Abcam (4 mentions)
Goat serum, by Santa Cruz Biotechnology (4 mentions)
Prolong gold antifade reagent, by Thermo Fisher Scientific (4 mentions)
Goat serum, by Beyotime (3 mentions)
Paraformaldehyde (pfa), by Merck Group (3 mentions)
Paraformaldehyde (pfa), by Polysciences (2 mentions)
Fluorescent microscope, by Leica camera (2 mentions)
Fluoromount g mounting medium, by Southern Biotech (2 mentions)
Alexa fluor 488 tagged anti chicken, by Thermo Fisher Scientific (2 mentions)
Alexa fluor 546 tagged anti mouse igg, by Thermo Fisher Scientific (2 mentions)
Fv10 asw 3.1 acquisition software, by Olympus (2 mentions)
Fv1000 confocal laser scanning microscope, by Olympus (2 mentions)
Anti gfp, by Abcam (2 mentions)
Goat serum, by Proteintech (2 mentions)
98 protocols using goat serum
1
Copper Nanoparticle Effects on Macrophage Mitochondria
2
Immunostaining of FLAG-tagged Neurons
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Cultured neurons were washed three times with 1 ml of prewarmed serum-free Neurobasal medium (Gibco, 21103049) supplemented with 4% B-27 (Gibco, 17504044) and 2 mM Glutamax (Gibco, 35050061), fixed in 4% paraformaldehyde (PFA) at room temperature for 15 min, and washed three times with phosphate-buffered saline (PBS). Cells were blocked with PBS containing 5% normal goat serum (Jackson ImmunoResearch, 005-000-121) at room temperature for 30 min and then stained with primary antibody against FLAG (DDDDK tag, Abcam, ab1162) at 1:200 dilution with 5% goat serum at 37°C for 1 hour and washed three times with PBS. Cells were then stained with Alexa Fluor 647 goat anti-rabbit secondary antibody (Abcam, ab150087) at 1:500 dilution with 5% goat serum at 37°C for 1 hour and washed three times with PBS. Cells were finally permeabilized in PBS containing 5% goat serum and 0.03% Triton X-100 at room temperature for 10 min, and mounted on slides using VECTASHIELD hardSet antifade mounting medium with DAPI (Vector Laboratories).
3
Immunofluorescent Staining of Citrullinated Histones
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After the NETs assay, the press-to-seal cover was gently lifted up, and the assay buffer was aspirated from the device wells. Then, the cells were fixed with 4% PFA (paraformaldehyde, 16%, methanol free, Polysciences, Warrington, PA) for 15 min at room temperature. Following the fixation, the cells were washed 3× with PBS at 5 min/wash. Later, the cells were blocked with 10% normal goat serum (Cell Signaling Technology, Danvers, MA) for 1 h at room temperature. The blocking buffer was then removed, and the cells were incubated in 4 μg mL−1 of the primary antibody (rabbit polyclonal anti-histone H3 (citrulline R2 + R8+ R17) antibody, Abcam, Waltham, MA) in 1% BSA + 1% goat serum in PBS buffer for 2 h at room temperature. The cells were then washed 3× with PBS at 5 min/wash. Finally, the cells were incubated in 20 μg mL−1 of the secondary antibody (goat anti-rabbit IgG H&L Alexa Fluor 647 pre-adsorbed, Abcam, Waltham, MA) in 1% BSA + 1% goat serum in PBS buffer for 1 h at room temperature (in the dark). The cells were then counter-stained with Hoechst solution (10 μg mL−1), followed by 2× washing with PBS at 5 min/wash and imaged.
4
Immunofluorescence Staining of Claudin 4 and Cryptosporidium parvum
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Confluent monolayers were fixed for 20min in 4% paraformaldehyde (Room temperature; RT), washed in PBS, then heated at 95°C for 10min with antigen retrieval buffer (10mM Tris Base, 1mM EDTA, 0.05% Tween, pH 9.5) and washed again in PBS. C. parvum infected cells were permeabilized with 0.2% Triton X-100 at RT for 3 min then washed in PBS. Cells were blocked with 20% goat serum (Abcam, Cambridge, UK) for 30min, incubated with 1:100 rabbit polyclonal anti-claudin 4 (antibodies from Abcam, Cambridge, UK) in 20% goat serum for 2h at RT, then washed in PBS and incubated with conjugated secondary antibody (anti-rabbit IgG H & L -AlexaFluor) for 1h at RT. Cells were mounted in Vectorshield including DAPI (Vector Laboratories, Peterborough, UK). For C. parvum staining, 1μg/ml -1 VVL (lectin of Vicia villosa, Vector laboratories, Peterborough, UK) in PBS with 1% Bovine serum albumin (BSA) was added to cells for 1h at RT, washed and incubated with 1μg/ml conjugated streptavidin CY3. After 10min, cells were mounted with vectorshield and DAPI. A Leica DM5000 upright epifluorescence microscope was used for images at 400x magnification. A Zeiss LSM710 point scanning confocal microscopy was used for representative confocal images at 400x magnification.
5
Cell Culture and PBMC Isolation Reagents
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Trypsin/EDTA and media used for cell culture (DMEM, DMEM-F12, IMDM, and RPMI 1640), as well as gradient cell separation medium used for PBMCs isolation (Lymphosep), were supplied by Biowest (Nuaillé, France). Normal-melting-point (NMP) agarose, low-melting-point (LMP) agarose, Triton X-100, 3-(4,5-dimethylthiazol-2-yl)-2,3-diphenyltetrazolium bromide (MTT), dimethyl sulfoxide (DMSO), 4,6-diamidino-2-phenylindole (DAPI), penicillin–streptomycin solution stabilized, fetal bovine serum (FBS), phytohaemagglutinin (PHA), phosphate-buffered saline (PBS), and N,N-Dimethylformamide (DMF) were purchased from Sigma-Aldrich (St Louis, MO, USA). Microplates, as well as 96-well and 12-well plates, were supplied by Thermo Fisher Scientific (Waltham, MA, USA). Other chemicals used in the experiments: fluoromount-G (Invitrogen, Carlsbad, CA, USA); bleomycin (TCI); SDS (ROTH); paraformaldehyde (Polysciences, Inc; 400 Valley Rd, Warrington, PA 18976, USA); goat serum (Abcam, Cambridge, UK); the primary antibody, anti-gamma-H2AX (phospho-Ser139) (Abcam, Cambridge, UK); the secondary Goat anti-Mouse IgG Cross-adsorbed antibody, Alexa Fluor 488 p (Invitrogen, Carlsbad, CA, USA).
6
Quantifying DNA Double-Strand Breaks
7
Immunocytochemistry of Adherent Cells
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Cells were placed in 24-well plates coated with poly-l -lysine and laminin.
After assuming their native shape, cells were fixed in 4% PFA for 40 min at RT, then
incubated in a mixture of 1% BSA (Abcam), 10% goat serum (Abcam), and 0.1% Tween-20 to
decrease the risk of unspecific binding and permeabilize the cell membranes if
necessary. Time of incubation and dilution depended on antibody characteristics (Table 1 ). After incubation with
primary antibody, secondary antibody was applied for 1 h (Alexa Fluor, 1:600) at RT [red
fluorochrome Alexa Fluor 594 nm, green fluorochrome Alexa Fluor 488 nm]. Nuclei were
stained with DAPI. Immunostainings were analyzed using a fluorescent microscope (Leica,
Wetzlar, Germany) equipped with a monocamera and LAS X software for picture
acquisition.
After assuming their native shape, cells were fixed in 4% PFA for 40 min at RT, then
incubated in a mixture of 1% BSA (Abcam), 10% goat serum (Abcam), and 0.1% Tween-20 to
decrease the risk of unspecific binding and permeabilize the cell membranes if
necessary. Time of incubation and dilution depended on antibody characteristics (
primary antibody, secondary antibody was applied for 1 h (Alexa Fluor, 1:600) at RT [red
fluorochrome Alexa Fluor 594 nm, green fluorochrome Alexa Fluor 488 nm]. Nuclei were
stained with DAPI. Immunostainings were analyzed using a fluorescent microscope (Leica,
Wetzlar, Germany) equipped with a monocamera and LAS X software for picture
acquisition.
8
Immunofluorescence Staining of Autophagy Markers
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LCL (1 × 106 cells/ml) were starved in EBSS with or without 10 nM or 100 nM bafilomycin A1 (Sigma) for 3 h at 37 °C and 5% CO2 before harvesting, when starvation was required. For LC3 staining, the cells were selectively permeabilized with 0.05% saponin prior to fixation. Cells were fixed in 4% formaldehyde for 20 min at room temperature, then permeabilized with 0.1% Triton X-100 and 2% goat serum (both Sigma-Aldrich) in PBS for 30 min on ice. After overnight incubation in 5% goat serum, cells were Fc receptor blocked (Human TruStain FcX, Biolegend) and incubated with 2 µg/ml rabbit anti-human CXORF21 (Atlas antibodies; HPA001185) and either 2 µg/ml mouse anti-human TLR7 (Novus Biologicals, NBP2–27332) or 40 µg/ml mouse anti-human LC3 (MBL, M152–3) in 5% goat serum for 1 h on ice. Following washing, cells were stained with goat anti-rabbit Alexa Fluor 488 (Abcam; ab150077) and goat anti-mouse Alexa Fluor 594 (Abcam; ab150116), both at 1:2000, in 5% goat serum for 30 min on ice. Cells were washed and mounted in ProLong™ Gold Antifade Mountant containing DAPI (Invitrogen).
9
Integrin β3 and CD31 Immunostaining
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Goat serum (Abcam, Cambridge, Massachusetts), rat anti-integrin β3 antibody (cat. no. 181720; BD Biosciences, Franklin Lakes, New Jersey), rat anti-CD31 antibody (1:100; cat. no. 551262; BD Biosciences), Cy3-conjugated goat anti-rat (1:100; cat. no. 115-165-003; Jackson ImmunoResearch Europe Ltd, Newmarket, the United Kingdom), goat anti-rat secondary antibodies (1:100; cat. no. 115-095-003; Jackson ImmunoResearch Europe Ltd), and glyceraldehyde phosphate dehydrogenase (GAPDH) were used as loading controls. The total protein level was normalized to the GAPDH protein level.
10
Immunofluorescence Imaging of Endothelial Cells
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After cancer cell rolling over the endothelium, the chips were fixed with 3.6% paraformaldehyde (Sigma-Aldrich) for 15 minutes at RT. The channel was washed three times with PBS, and cells were permeabilized with 0.1% Triton X-100 for 10 minutes on ice followed by three washes with PBS. Then, they were incubated with 20% goat serum (Sigma-Aldrich) for 30 minutes and then incubated for 3 hours with mAb mouse Ve-cadherin (Abcam, 1:100) in 10% goat serum at 4°C. After several washes with PBS, cells were incubated 50 minutes with the mAb goat FITC conjugated (Abcam, 1:500) and Hoechst. After washing, chips were imaged with the Nikon A1R-A1 confocal microscope.
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