Application
Immunohistochemistry (Frozen sections)
Sample
Mouse Tissue sections (thymus, eye)
Specification
thymus, eye
Fixative
Paraformaldehyde
Permeabilization
Yes - tritonX-100 10min
Blocking step
BSA as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 1% · Temperature: 25°C
Other product details
Dilution
1/500
Incubation time
2 hour(s) and 30 minute(s) · Temperature: 25°C · Diluent: 1% BSA in PBS- 0.1% Tween-20
Secondary antibody
Secondary antibody
None used
Additional data
Additional Notes
I used this antibody in my in situ hybridization experiments with DiG labelled RNA probes, instead of the "standard" Roche anti-DiG-AP Fab fragments to increase the sensitivity of my method since I could now use Vector's ABC system. Unfortunately, The results are disappointing... In a side to side experiment with Roche anti-DiG-AP, this antibody gave more background, less intense staining and is more complicated to use because of the use of the avidin-biotin complex. Unless I had a bad batch I cannot explain the poor results in any other way except that the antibody is useless for the detection of digoxigenin labelling of nucleotides. I do not recommend its use for ISH.
Abcam response
Thank you for your valuable feedback. Although we do not have previous ISH reports for this product, the same clone is available as ab6212, HRP-conjugated. Two recent publications include ISH data obtained with ab6212.(https://www.abcam.com/Digoxigenin-antibody-HRP-21H8-HRP-ab6212.html). The clone is also available in an unconjugated format as ab420: https://www.abcam.com/Digoxigenin-antibody-21H8-ab420.html
Your comparison with the Roche product suggests that, for your samples and protocol, there is an advantage to using Fab fragments versus a whole antibody such as ab419. High background after an avidin-biotin amplification may be due to endogenous biotin. We recommend a block for this consisting of free avidin followed by free biotin.
Your comparison with the Roche product suggests that, for your samples and protocol, there is an advantage to using Fab fragments versus a whole antibody such as ab419. High background after an avidin-biotin amplification may be due to endogenous biotin. We recommend a block for this consisting of free avidin followed by free biotin.
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.
DR. Lampros Panagis
Verified customer
提交于 Feb 10 2011