Application
Immunohistochemistry (Frozen sections)
Blocking step
BSA as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: RT°C
Sample
Mouse Tissue sections (Brain)
Specification
Brain
Permeabilization
Yes - 0.1% Triton-X (in PBS)
Fixative
Paraformaldehyde
Other product details
Incubation time
12 hour(s) and 0 minute(s) · Temperature: 4°C · Diluent: 5% BSA in PBS
Dilution
1/100
Secondary antibody
Name
Non-Abcam antibody was used: Invitrogen - Alexa Fluor® 488 Goat Anti-Rabbit IgG
Host species: Goat
Clonality: Polyclonal
Conjugation: Alexa Fluor® 488
Host species: Goat
Clonality: Polyclonal
Conjugation: Alexa Fluor® 488
Dilution
1/500
Additional data
Additional Notes
Fixative: 4% PFA (in PBS)
The protocol included a postfixation step (4% PFA) before the permeabilization step.
Secondary antibody was diluted in PBS.
The brain tissue was collected from E13.5 murine embryos.
The picture shows a closeup of the lateral cortex (magnification: 40X).
Specific staining could be detected only in one experiment. The result could never be replicated in subsequent experiments.
Abcam response
We are working with the customer to understand why the results were not repeatable. For PFA or formalin-fixed samples paraffin-embedded tissue, an antigen retrieval step is recommended, and antigen retrieval (heat-mediated) may be necessary for consistent results from frozen tissue.
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
提交于 Jun 12 2013