Application
Immunohistochemistry (Frozen sections)
Sample
Mouse Tissue sections (5um Aorta Cryosections)
Permeabilization
Yes - 0.1% Triton-X 100 for 20min
Specification
5um Aorta Cryosections
Blocking step
BSA as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 0.5% · Temperature: 22°C
Fixative
Paraformaldehyde
Other product details
Incubation time
1 hour(s) and 0 minute(s) · Temperature: 22°C · Diluent: TBS with 0.5% BSA
Dilution
1/400
Secondary antibody
Dilution
1/1000
Name
Non-Abcam antibody was used: Donkey anti-Rabbit A555
Host species: Donkey
Clonality: Polyclonal
Conjugation: Alexa Fluor® 555
Host species: Donkey
Clonality: Polyclonal
Conjugation: Alexa Fluor® 555
Additional data
Additional Notes
Figure 2: Abcam antibody 60946 does not reliably detect insulin receptor phosphorylation in ex vivo murine aortas. BALB/c mice (food-deprived for 6 hrs) were sacrificed by cardiac puncture and the right ventricle was perfused with heparin (10 u/mL) to prevent blood clotting. Murine aortas were then isolated. Following a 2-hr ex vivo tissue serum-deprivation in DMEM with 0.1% BSA, aortas were perfused at 300 µL/min with 100 nM of insulin for 10 min. The tissue was fixed with manual perfusion of 4% PFA for 3 min, followed by a 30 min incubation in 4% PFA. Tissues were mounted in OCT and frozen at -80C. 5 µm cross-sectional cryosections of the aorta were mounted on glass slides. Slides were allowed to air-dry overnight. Tissues were rehydrated for 10 minutes in TBS, and permeabilized in 1% SDS for 3 min before blocking in 0.5% BSA for 1 hr at RT. The aorta cryosections were incubated for 1 hr at RT with abcam60946 (1:400), followed by a 1 hr incubation with a fluorescent secondary (A555, red). DaKo mounting media with DAPI was used to adhere a coverglass over the tissue section and it was imaged using spinning disk confocal microscopy at 60X magnification. 8 aortic rings from each treatment (control and insulin treated) were stained, and 3 images were taken from each aortic ring. Insulin receptor signal (A555,red) was normalized to smooth muscle staining (A488, green) for each image. Each data point represents the average of three images taken per aortic ring. Error bars are SD.
Abcam response
We would like to thank the customer for submitting this valuable feedback in form of an AbReview. The customer took part in our AbTrial program to test this antibody without financial risk in an so far for this product untested application.
We would be very happy to receive additional feedback from other customers as well that have tested this antibody in Immunohistochemistry on frozen sections.
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.
Victoria Tokarz
Verified customer
提交于 Jul 14 2016