HeLa (cervical cancer) was originally purchased from ATCC and MEF cells were a gift from D.J. Kwiatkowski (Harvard Medical School). Cells were not authenticated recently but tested negative for mycoplasma contamination. Both cells were maintained in Dulbecco's Modified Eagle's Medium (DMEM) with 10% fetal bovine serum (FBS). Antibodies used in the experiments are listed below: anti-YTHDF2 (Proteintech 24744-1-AP, 1:1000 WB, 1:600 IF); anti-Hsp70 (Stressgen SPA-810, 1:1000 WB); anti-FTO (Phosphosolutions 597-Fto, 1:1000 WB, 1:600 IF); anti-METTL3 (Abnova H00056339-B01P, 1:1000 WB, 1:600 IF); anti-METTL14 (sigma HPA038002, 1:1000 WB, 1:600 IF); anti-WTAP (Santa Cruz sc-374280, 1:1000 WB, 1:600 IF); anti-m6A (Millipore ABE572, 1:1000 m6A immunoblotting); Alexa Fluor 488 donkey anti-mouse secondary antibody (Invitrogen A21202. 1:600 IF); Alexa Fluor 546 donkey anti-rabbit secondary antibody (Invitrogen A10040, 1:600 IF).
Alexa fluor 546 donkey anti rabbit secondary antibody
Manufactured by Thermo Fisher Scientific
Sourced in Italy
Alexa Fluor 546 donkey anti-rabbit secondary antibody is a fluorescently-labeled antibody that binds to rabbit primary antibodies. It is designed for use in immunofluorescence applications.
Automatically generated - may contain errors
Lab products found in correlation
Abe572, by Merck Group (2 mentions)
H00056339 b01p, by Merck Group (2 mentions)
Spa 810, by PhosphoSolutions (2 mentions)
Hpa038002, by Santa Cruz Biotechnology (2 mentions)
Sc 374280, by Merck Group (2 mentions)
Alexa fluor 488 donkey anti mouse secondary antibody, by Thermo Fisher Scientific (2 mentions)
Anti eif4a1, by Abcam (1 mentions)
Anti puromycin, by Developmental Studies Hybridoma Bank (1 mentions)
Anti β actin, by Merck Group (1 mentions)
4 protocols using alexa fluor 546 donkey anti rabbit secondary antibody
1
HeLa and MEF Cells for m6A Research
2
HeLa and MEF Cells for m6A Research
3
Immunoblotting of Eukaryotic Translation Initiation Factors
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MEF, HEK293, and HEK293-Kb cells were maintained in Dulbecco’s Modified Eagle’s Medium (DMEM) with 10% fetal bovine serum (FBS). Antibodies used in the immunoblotting are listed below: anti-eIF4A1 (Abcam ab31217), Anti-eIF4A2 (Life technologies, PA585142), anti-puromycin (Developmental Studies Hybridoma Bank PMY-2A4), anti-β-Actin (sigma A5441), Alexa Fluor 546 donkey anti-mouse secondary antibody (Invitrogen A10036) and Alexa Fluor 546 donkey anti-rabbit secondary antibody (Invitrogen A10040).
4
Immunofluorescence Staining of 2D and 3D Organoids
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After washing the slides with 1X PBS to remove OCT for 3D organoids, the 2D organoids were openly seeding in six wells pretreated with Matrigel diluted 1:40 in PBS; the tissues were permeated with 0.15% Triton/1X PBS for 15 min at room temperature. The cells were washed 3 times with 1X PBS for 10 min each time. The slides were blocked with 3% BSA/1X PBS (blocking solution) or 10% FBS/1X PBS for 1 h at room temperature. The primary antibody anti-rabbit anti-cytokeratin (cat. Z0622, Dako Carpinteria, CA, USA) was diluted in blocking solution overnight at 4 °C in a humidified chamber. Then the cells were washed 3 times with 1X PBS for 10 min each time. Alexa Fluor 546 donkey anti-rabbit secondary antibody (A10040 Invitrogen, Milan, Italy) was added to the organoids. The cells were then washed 3 times with 1X PBS for 10 min each time, mounted with Mowiol (cat. 475904-M Sigma-Aldrich, Milan, Italy) and observed under a confocal microscope (Zeiss LSM 510, Jena, Germany) [18 (link)].
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