Product Review: VISA antibody-N-terminal region (ARP49558_P050) in 293T cells transfected with 3FLAG-VISA using IHC

Product Page for VISA antibody-N-terminal region (ARP49558_P050)

Researcher: Dr. Safia Deddouche, London Research Institute
Application: IHC
Species+tissue/cell type:293T cells transfected with 3FLAG-VISA
Primary antibody dilution: 1:500
Secondary antibody: Anti-rabbit-Alexa 488
Secondary antibody dilution:1:500

Questionnaire:
How do Aviva’s reagents play a role in your experimental goals?
How would you rate this antibody on a scale from 1-5 (5=best) and why? 4-staining is good little background and reasonable affinity
How did you store the antibody after re-suspension? At -20C and an aliquot at 4C for few weeks
Sample Description (please include species type and tissue/cell type): Hek 293T cells either non transfected or transfected with a plasmid encoding either 3Flag MDA5 or 3Flag MAVS.
Please explain fixation solution/method used (formalin, periodate-lysine-paraformaldehyde, acetone, etc.)? 4% PFA for 10min at RT
How many different experimental trials were conducted using the antibody sample? 1
Experimental Procedure/Protocols: 293T cell Lipofectamine 2000 ( Invitrogen) transfected with 3Flag-VISA or MDA5
incubation o/n
wash cells once PBS
Add Hoescht 3332 (nuclear stain) 20uM and MitoTracker Far RED (Molecular probe) 20uM diluted in PBS for 30min at 37C.
wash cells once PBS
Fixation 15min at 37C in 4% PFA
Wash twice PBS
Permeabilize 10min PBS+ 0.1% Triton x100 at room temperature
Block with PBS + 0.1% Triton x100 + 3% BSA for 1h at room temperature
Incubate with anti-VISA (ARP49558_P050 – VISA) or anti-MDA5 (ARP36486_P050) d 1/500 in PBS + 0.1% Triton x100 + 0.3% BSA for 18h at 4C
Wash twice PBS
Incubate with anti-Rabbit Alexa 488 (Molecular Probes) d 1/500 in PBS + 0.1% Triton x100 + 0.3% BSA for 18h at 4C 2h at room temperature.
Wash twice PBS
Mount in Fuoromount-G (SoutherBiotech)
Image with confocal LSM 510.

 

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