Immunofluorescence was implemented as previously reported [13 ]. Briefly, BGC823 ISL1‐knockdown, GLUT4‐knockdown, and scramble cells were fixed in 4% paraformaldehyde for 20 min, cultured in blocking buffer, then incubated with primary antibodies against GLUT4 and ISL1, followed by incubation with Alexa Flour donkey anti‐rabbit and donkey anti‐mouse antibodies (Invitrogen). The 4′,6‐diamidino‐2‐phenylindole 4 (DAPI, Invitrogen) binding to DNA can be used to observe nuclear condensation [35 ]. The images were obtained by laser scanning confocal microscope LSM 780 with Zen software under a 63 × oil‐immersed lens (Carl Zeiss, Toronto, ON, Canada). The details of the antibodies used are listed in Supplementary Table S3 .
Alexa flour donkey anti rabbit and donkey anti mouse antibodies
Manufactured by Thermo Fisher Scientific
Alexa Fluor donkey anti-rabbit and donkey anti-mouse antibodies are secondary antibodies used in immunodetection and imaging applications. They are conjugated with Alexa Fluor dyes, which provide bright and photostable fluorescent labels. These antibodies are designed to bind to primary antibodies raised in rabbit or mouse, allowing for the detection and visualization of target proteins or other biomolecules.
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2 protocols using alexa flour donkey anti rabbit and donkey anti mouse antibodies
1
Immunofluorescence Analysis of GLUT4 and ISL1
2
Immunofluorescence-based Analysis of USP15, HDKC1, and IGF2BP3
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Immunofluorescence was implemented as previously reported. Briefly, USP15 knockdown or negative control GC cells were fixed in 4% paraformaldehyde for 20 min, cultured in blocking buffer, then incubated with primary antibodies against USP15, HDKC1, and IGF2BP3, followed by incubation with Alexa Flour donkey antirabbit and donkey anti-mouse antibodies (Invitrogen). The DAPI (Invitrogen) binding to DNA can be used to observe nuclear condensation. The images were obtained by laser scanning confocal microscope LSM 780 with Zen software under a 63 × oil-immersed lens (Carl Zeiss, Toronto, ON, Canada).
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