Animals were boiled in 1× SDS loading buffer (65mM Tris pH 6.8, 10% glycerol, 2% SDS, 2mM PMSF, 1× Halt Protease Inhibitor (Thermo), 10% 2-mercaptoethanol) and run on a 7.5% Criterion TGX gel (Bio-Rad Laboratories, Inc.). Protein was transferred to a low background fluorescence PVDF membrane (Millipore). The membrane was blocked in 3% nonfat milk in 1× PBST with 250mM NaCl. The 5–6 antibody was used at a 1:5000 dilution to detect myo-3, and 5–8 antibody used at a 1:5000 dilution to detect unc-5452 . The 5–6 and 5–8 monoclonal antibodies were produced previously by purification of endogenous myosin proteins. Secondary antibody staining was performed with 1:500 Cy3-conjugated affiniPure goat anti-mouse (Jackson Immunoresearch). Imaging was done on a Typhoon Trio (Amersham Biosciences), and quantification done in ImageJ. For the lower blot of Fig 3D , lysates were made from multiple animals, and serial dilutions done to titrate the number of animals per lane.
Cy3 conjugated affinipure goat anti mouse
Manufactured by Jackson ImmunoResearch
Sourced in United States
Cy3-conjugated affiniPure goat anti-mouse is a secondary antibody used for detection and visualization in various immunoassays and imaging techniques. The antibody is conjugated with the Cyanine 3 (Cy3) fluorescent dye, which emits light in the red-orange region of the visible spectrum when excited. This product is suitable for applications that require specific detection of mouse primary antibodies.
Automatically generated - may contain errors
Lab products found in correlation
Pvdf membrane, by Merck Group (1 mentions)
Halt protease inhibitor, by Thermo Fisher Scientific (1 mentions)
Criterion tgx gel, by Bio-Rad (1 mentions)
Typhoon trio, by Cytiva (1 mentions)
Tcs sp8, by Leica (1 mentions)
Cd11b, by BD (1 mentions)
Fitc conjugated affinipure goat anti rabbit igg, by Jackson ImmunoResearch (1 mentions)
F4 80, by Santa Cruz Biotechnology (1 mentions)
Zippy 1, by Adipogen (1 mentions)
2 protocols using cy3 conjugated affinipure goat anti mouse
1
Western Blot Analysis of Myosin Proteins
2
Immunofluorescence Analysis of Liver Samples
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Liver samples were fixed in 4% paraformaldehyde and embedded in Tissue Tek OCT compound. Frozen sections of 5 μm were used for immunofluorescence. Liver sections were blocked with 2% BSA, and then incubated with anti-ZBP1 (Adipogen, Zippy-1, AG-20B-0010, 1:100), anti-mouse MLKL (phospho S345, Abcam, ab196436, 1:50), anti-human MLKL (phospho S358, Abcam, ab187091, 1:50), CD68 (Santa Cruz, sc-9138, 1:100), CD86 (Santa Cruz, sc-28347, 1:100), CD11b (BD, 550282, 1:100), F4/80 (Santa Cruz, sc-59171, 1:100). FITC-conjugated affinipure goat anti-rabbit IgG, Cy3-conjugated affinipure goat anti-mouse, and Cy5-conjugated affinipure goat anti-rat IgG (1:200, Jackson Immunoresearch, PA, USA) were used as secondary antibodies. The sections of the livers were subjected to TUNEL analysis by using an FITC-TUNEL apoptosis detection kit (Yeasen, Shanghai, China) according to the manufacturer’s protocol. Finally, the sections were stained with DAPI and observed under a confocal microscope (Leica TCS SP8, Leica Microsystems, Mannheim, Germany).
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