For both IHC and immunofluorescence (IF), 4-μm paraffin sections were deparaffinized with xylene. Slides were rehydrated using decreasing concentrations of ethanol. Antigen retrieval was performed at 121°C with the slides in either EDTA Ph9 or Citrate Ph6. Primary antibodies for GFPT2 (1:500; ph9; ab190966; Abcam), PU.1 (1:50; ph6; 554268; BD Biosciences), CD68 mouse (1:200; ph9; ab955; Abcam), CD68 rabbit (1:200; ph9; 76437S; Cell Signaling Technology), MMP9 (1:1,000; ph9; HPA001238; Millipore Sigma), YAP1 (1:1,200; ph6; sc-101199; Santa Cruz), desmin (1:40; D33 Dako), PDPN (D2–40 Dako) and Ki67 (1:50; ph9; Cell Signaling Technology) were used. For IHC immunodetection was completed using the Vectastain ABC kit (Vector Laboratories) and DAB chromogen (Abcam), according to the manufacturer's specifications. For IF a secondary mouse or rabbit Alexa488, Alexa555 or Alexa657 labeled antibodies were used (ThermoFisher). IHC and IF sections were scanned using the Keyence Fluorescence Microscope (BZ-X; Keyence) with the 40x lens, either using the fluorescent filter cube or the brightfield setting.
Ab190966
Manufactured by Abcam
Ab190966 is a monoclonal antibody that binds to the Human CD4 antigen. The antibody is produced in mouse and is purified from cell culture supernatant.
Automatically generated - may contain errors
Lab products found in correlation
Pierce bca protein assay kit, by Thermo Fisher Scientific (3 mentions)
Alexa 555, by Thermo Fisher Scientific (1 mentions)
Dab chromogen, by Abcam (1 mentions)
Sc 101199, by Santa Cruz Biotechnology (1 mentions)
Alexa 488, by Thermo Fisher Scientific (1 mentions)
Vectastain abc kit, by Vector Laboratories (1 mentions)
Ab955, by Abcam (1 mentions)
Ab40772, by Abcam (1 mentions)
Ab2739, by Abcam (1 mentions)
Ab8226, by Abcam (1 mentions)
Ab92547, by Abcam (1 mentions)
Ab125069, by Abcam (1 mentions)
G 21234, by Thermo Fisher Scientific (1 mentions)
G 21040, by Thermo Fisher Scientific (1 mentions)
Ecl solution, by Merck Group (1 mentions)
T9026, by Merck Group (1 mentions)
Chemidoc mp imaging system, by Bio-Rad (1 mentions)
Trans blot turbo transfer system, by Bio-Rad (1 mentions)
3 protocols using ab190966
1
Immunohistochemistry and Immunofluorescence Protocols
2
Western Blot Analysis of Cellular Markers
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Western blotting was performed using antibodies against GFPT2 (1:1000, ab190966, Abcam), YBX1 (1:1000, A3534, ABclonal), IL-18 (1:2000, 10663-1-AP, Proteintech), O-GlcNAcylation (1:1000, ab2739, Abcam), OGT (1:1000, #24083, Cell Signaling Technology), E-cadherin (1:1000, ab40772, Abcam), vimentin (1:1000, ab92547, Abcam) and beta actin (1:1000, ab8226, Abcam).
3
Protein Expression Analysis by Western Blot
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Protein concentration of cell lysates was determined using the Pierce TM BCA protein assay kit according to manufacturer's instructions (ThermoFisher Scientific). Samples were adjusted in 5xLDS sample buffer containing 50 mM DTT. After boiling and a sonication step, equal protein amounts were subjected to SDS-PAGE and blotted on a nitrocellulose membrane using the Trans-Blot
Turbo Transfer system (BioRad). All antibodies were used in 5% low-fat milk or 5% BSA in TBS-Tween. After incubation with HRP-conjugated secondary antibody, the blot was developed using ECL solution (Merck Millipore) on a ChemiDoc MP Imaging System (BioRad).
The following antibodies were used in this study: GFAT1 (rb, EPR4854, Abcam ab125069, 1:1000), GFAT2 (rb, Abcam, ab190966, 1:5000), O-Linked N-Acetylglucosamine Antibody (ms, clone RL2, MABS157, Merck, 1:1000), AMDHD2 (ms, S6 clone, in-house produced, 1:500), a-TUBULIN (ms, clone B-5-1-2, Sigma T9026, 1:5000), rabbit IgG (gt, HRP-conjugated, G21234, Thermo
Fisher,1:5000), and mouse IgG (gt, HRP-conjugated, G21040, Thermo Fisher, 1:5000).
Turbo Transfer system (BioRad). All antibodies were used in 5% low-fat milk or 5% BSA in TBS-Tween. After incubation with HRP-conjugated secondary antibody, the blot was developed using ECL solution (Merck Millipore) on a ChemiDoc MP Imaging System (BioRad).
The following antibodies were used in this study: GFAT1 (rb, EPR4854, Abcam ab125069, 1:1000), GFAT2 (rb, Abcam, ab190966, 1:5000), O-Linked N-Acetylglucosamine Antibody (ms, clone RL2, MABS157, Merck, 1:1000), AMDHD2 (ms, S6 clone, in-house produced, 1:500), a-TUBULIN (ms, clone B-5-1-2, Sigma T9026, 1:5000), rabbit IgG (gt, HRP-conjugated, G21234, Thermo
Fisher,1:5000), and mouse IgG (gt, HRP-conjugated, G21040, Thermo Fisher, 1:5000).
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