Application
Flow Cytometry
Sample
Mouse Cell (Fluorescent assisted cell sorting)
Specification
Fluorescent assisted cell sorting
Preparation
Cell harvesting/tissue preparation method: Cortex digested with Accutase, debris filtered via percoll gradient, cells strained through a 40uM filter and then stained with 1Ab
Sample buffer: RNAse free PBS
Sample buffer: RNAse free PBS
Fixation
No fixation
Permeabilization
No
Gating Strategy
neuronal cells were gated
Other product details
Incubation time
30 minute(s) · Temperature: 4°C · Diluent: PBS
Secondary antibody
Name
Non-Abcam antibody was used: Jackson Dylite 488 Secondary Ab
Host species: Donkey
Clonality: Polyclonal
Conjugation: Alexa Fluor® 488
Host species: Donkey
Clonality: Polyclonal
Conjugation: Alexa Fluor® 488
Dilution
1/200
Additional data
Additional Notes
This Ab was used to separate neuronal populations from non-neuronal populations in the adult mouse cortex via FACs for the purpose of RNA extraction. The antibody showed no specificity for neurons only, as the entire brain homogenate cell population stained positive for NrCaM a marker which should only be on the neuronal surface (hence no need for fixation or permeabilization). Secondary specificity was confirmed as no staining was observed in the absence of the primary Ab. Thus the problem arose from non-specific binding of the primary Ab.
Discount Code:100%ABR-T7ABZ
Discount Code:100%ABR-T7ABZ
Abcam response
Our Scientific Support team is currently investigating this negative Abreview, and we will resolve the issue in accordance with our Abpromise.
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 Mar 20 2012