Application
Immunocytochemistry/ Immunofluorescence
Sample
Human Cell (293 human embryonic kidney)
Permeabilization
Yes - 0,5%(w/v) saponin
Specification
293 human embryonic kidney
Blocking step
Serum as blocking agent for 2 hour(s) and 0 minute(s) · Concentration: 10% · Temperature: 23°C
Fixative
Paraformaldehyde
Other product details
Incubation time
2 hour(s) and 0 minute(s) · Temperature: 23°C · Diluent: 0,5%(w/v) saponin
Dilution
1/200
Secondary antibody
Dilution
1/400
Name
Non-Abcam antibody was used: goat anti mouse AlexaFluor488
Host species: Goat
Clonality: Polyclonal
Conjugation: Alexa Fluor 488
Host species: Goat
Clonality: Polyclonal
Conjugation: Alexa Fluor 488
Additional data
Additional Notes
Cells were plated on an uncoated cover slip.
One day after plating cells were washed twice with PBS and fixed with 4% Paraformaldehyde (including 4% sucrose) for 15 minutes at room temperature. Cells were then permeabilized at 4¯C for 45min with 0.5% saponin in PBS.
Blocking was carried out with 10% fetal calf serum in PBS (+0.5% saponin) during first and second antibody incubation.
After additional washing cover splips were mounted on glass slides with ProLong Gold antifade reagent with DAPI (Invitrogen). The antibody does not work on PFA-fixated cells, nor on MetOH-fixated cells. Tested dilutions ranged from 1:50 to 1:200
Abcam response
Thank you for submitting an Abreview for ab52901.
We greatly appreciate your feedback and strive to publish both negative and positive reviews, rewarding each with Abpoints. We would like to make a few suggestions taken from comparing your protocol to that used successfully by the laboratory for testing.
Blocking buffer should be 10% serum from host of secondary antibody. Permeabilization was not used with this antibody. We use Tween 20 to reduce surface tension in washing solutions, but this detergent does not permeabilize fixed cells.
Please let us know if you require additional assistance. We offer an Abpromise guarantee to replace or refund the product if it does not perform as stated on the datasheet provided the antibody was received within the last 120 days.
The reviewer received a reward from Abcam’s Loyalty Program in thanks for submitting this Abreview and for helping the scientific community make better-informed decisions.
Abcam user community
Verified customer
投稿 Aug 26 2010