Application
Immunocytochemistry
Sample
Rat Cultured Cells (Astrocytes)
Permeabilization
Yes - Triton-X100
Specification
Astrocytes
Blocking step
Serum as blocking agent for 30 minute(s) · Concentration: 50% · Temperature: 24°C
Fixative
Formaldehyde
Other product details
Incubation time
24 hour(s) and 0 minute(s) · Temperature: 4°C
Dilution
1/500
Secondary antibody
Dilution
1/1000
Name
Non-Abcam antibody was used: AlexaFluor555
Host species: Goat
Clonality: Polyclonal
Conjugation: Alexa Fluor 555
Host species: Goat
Clonality: Polyclonal
Conjugation: Alexa Fluor 555
Additional data
Additional Notes
We used this antibody, anti-GFAP, and anti-S100beta, and this antibody was the only one of the three that did not stain our rat astrocyte cultures.
CUSTOMER'S RESPONSE TO ABCAM REPLY:
I wanted to address the Abcam response to my review of this antibody. The block that we use is from LiCor (Odyssey block has some fish serum in it), and it is actually supposed to be used full strength. We used it at 50% strength to save on cost, and this dilution works well with our other antibodies. Second, our staining with secondaries at the 1:1000 dilution is usually excellent, with a high signal to noise ratio. Secondaries from Invitrogen lead to more background if used at 1:200 because they are so bright, so I would not recommend that. In general, it is best to use lower dilutions to maximize the signal to noise ratio. In other words, the dilution of the block and the secondary are not the explanations for the lack of staining. As a positive control, we stained the same cultures for GFAP in parallel and that worked well.
Abcam response
As we have not tested ab87117 in cultured cells (ICC), it is possible that the antibody is not suitable for this application or that the protocol must be altered. We would recommend using a more dilute blocking serum (10% serum in PBST) and a more concentrated primary antibody solution (1:200 in PBST) to see if the results improve.
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.
Rehana Leak
Verified customer
投稿 Jul 01 2011