Question (52190) | Anti-LMX1b antibody (ab66941)

Go to datasheet (ab66941)

Question

Embryonic midbrain 30 µm sections fixed with 4% paraformaldehyde. Blocking and permeabilization solution: we have tried 2 methods 1. 10% NGS, 0.3% Triton X-100 in PBS for 1 hr 2. HCL 1N 15 min, 3 PBS washes (5 min each), Sodium borate buffer 0.1 M 10 min and then 10% NGS, 0.3% Triton X-100 in PBS+BSA 0.1% for 1 hr Primary antibody incubation overnight in 10% NGS in PBS+BSA 0.1% Dilutions tested: 1:100, 1:500 and 1:1000 3 washes with PBS+BSA 0.1% Secondary antibody alexa 488 or 568 diluted 1:500 in PBS+BSA 0.1% 2 hrs at room temperature. 3 washes with PBS+BSA 0.1% With these protocols we have not been able to see any staining with both antibodies

Answer

Thank you for sending your protocol. I am sorry to see that this antibody and the LMX1a antibody ab31006 are both giving negative IHC results.

I think your protocol is good and I would expect to see a positive result for ab66941, though we have had mixed reports, as you can see from the customer reviews shown on the datasheet. There is a question of specificity according to one of the reviewers but the western blot data suggests the antibody is specific. For both reviewers, however, at least some staining was evident..

Can you please confirm that the species of your sample is mouse? Are you confident that the secondary antibody will recognize rabbit IgG, and that it is effective with other antibodies?

The LMX1a antibody was found some years ago to be ineffective for IHC. We unfortunately do not have another antibody against this protein that has been shown to be capable of staining tissue sections.

Can you please tell me approximately when you placed your order for the LXM1b antibody? I am willing to discuss replacing it,, as IHC of mouse tissue is covered by our guarantee, though our choices of good alternatives is very limited.

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