SARS-CoV-2–infected samples were fixed with 10% formalin for 1 hour at room temperature, as recommended by C-CL3 safety protocol. Subsequently, samples were washed three times with PBS, and then blocked with PBS containing 10% FBS and 0.1% Triton X-100, at 4°C overnight. The tissues were then incubated with primary mouse monoclonal anti-human cardiac troponin T (Thermo Fisher Scientific) or mouse monoclonal anti-human CD3 (Thermo Fisher Scientific), at 4°C, overnight. The samples were then washed three times with PBS for 15 min, incubated with anti-mouse Alexa Fluor 488 (Life Technologies) and anti-mouse Alexa Fluor 647 (Abcam) secondary antibody, overnight, at 4°C, and then rinsed three times with PBS. For PBMC identification, it was tagged with CellTracker dye CM-DiI according to the manufacturer’s protocol. Immunofluorescence images were captured with a confocal fluorescence microscope (Nikon A1R).
Anti mouse alexa fluor 647
Manufactured by Abcam
Anti-mouse Alexa Fluor 647 is a fluorescent dye conjugated to an anti-mouse secondary antibody. It is used for the detection and visualization of mouse primary antibodies in various immunoassays and imaging applications.
Automatically generated - may contain errors
Lab products found in correlation
Anti mouse alexa fluor 488, by Thermo Fisher Scientific (1 mentions)
Elyra ps 1 lsm 780 microscope, by Zeiss (1 mentions)
Alexa fluor 647, by Abcam (1 mentions)
Triton x, by Merck Group (1 mentions)
Fetal calf serum (fcs), by Thermo Fisher Scientific (1 mentions)
Dako mounting medium, by Agilent Technologies (1 mentions)
α mhc, by Abcam (1 mentions)
Cold water fish skin gelatin, by Merck Group (1 mentions)
Sp5 laser scanning confocal microscope, by Leica (1 mentions)
Saponin, by Merck Group (1 mentions)
Paraformaldehyde (pfa), by Merck Group (1 mentions)
Dna dye dapi, by Thermo Fisher Scientific (1 mentions)
Rabbit anti mpo, by Abcam (1 mentions)
Tcs sp2 aobs apparatus, by Leica (1 mentions)
Prolong gold anti fade media, by Thermo Fisher Scientific (1 mentions)
3 protocols using anti mouse alexa fluor 647
1
Immunofluorescence Staining of SARS-CoV-2 Samples
2
Immunofluorescence Staining of Cardiomyocytes
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Cells on coverslips were fixed in 4% paraformaldehyde (Sigma) for 1 hr at 4°C, permeabilized with 0.1% Triton-X (Sigma), and blocked with 0.2% cold-water fish skin gelatin (Sigma)/10% FCS (GIBCO). All antibodies were incubated in blocking solution plus 0.01% saponin (Sigma), primary antibodies (TBX3, 1:200 [Abnova]; α-MHC, 1:200; [Abcam]; HCN4, 1:100 [Alomone Labs]; Connexin 30.2, 1:200 [Abcam]; and Connexin 45, 1:200 [Abcam]) for 1 hr at RT, secondary antibodies (anti-sheep Alexa Fluor 633, 1:500 [Life Technologies]; anti-mouse Alexa Fluor 647, 1:500 [Abcam]; and anti-rabbit Alexa Fluor 647, 1:500 [Abcam]) for 45 min at RT with additional phalloidin (1:500; Enzo Life Science) and bisBenzimide (DAPI, 1:2,000; Sigma). The coverslips were mounted with Dako mounting medium (DAKO) to glass slides and analyzed by fluorescence microscopy using a Leica SP-5 confocal laser scanning microscope (Leica Microsystems) or an ELYRA PS.1 LSM 780 microscope (Carl Zeiss).
3
Immunohistochemical Staining of PsA and OA Synovia
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Three-μm-thick sections in paraffin of human PsA and OA synovia were stained after deparaffination in xilene (5 min, two times), followed by passages in: absolute ethanol (3 min), 95% ethanol in water (3 min), 80% ethanol in water (3 min), 70% ethanol in water, and antigen retrival (5 min at 95°C in 10 mM sodium citrate, pH 6.0). Slides were saturated with blocking buffer (PBS, 0.05% tween 20, 4% BSA) for 1 hour at room temperature. Specimens were stained with a polyclonal rabbit anti-LL37 (Innovagen), rabbit anti-MPO (Abcam), mouse anti-MxA (Novus Bio), monoclonal mouse anti-LL37 (Mab137), polyclonal rabbit anti-human C9 (ATLAS). The following antibodies were used: donkey anti-rabbit IgG AlexaFluor-568 or-647, anti-mouse AlexaFluor-647 and an anti-goat AlexaFluor-488 (Abcam). After washing, slides were mounted in Prolong Gold anti-fade media containing a DNA dye (DAPI) (Molecular Probes). CLSM observations were performed with a Leica TCS SP2 AOBS apparatus, using a 63x/1.40 NA oil objective. Acquisition of images was performed by a Leica confocal software 2.6 (Leica, Germany).
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