Question (69922) | Anti-Cardiac Troponin I antibody (ab47003)

Go to datasheet (ab47003)

Question

Dear Tech Support Team,
Please read below and see the attached images (the troponin is green, cd31is pink) and advise to interpret the results.
Thanks in advance for your assistance.
Antibody code: ab47003,24590
Batch number:
General Information:
Antibody storage conditions (temperature/reconstitution etc) as stated in the date sheet. -20 in diluents
Description of the problem (high background, low signal, non-specific staining etc.)
non-specific staining and the staining itself is not like in the pictures in abcam webpage
Sample (Species/Tissue/Cell Type/Cell Line etc.) cells (cardiomyocytes and HUVEC on a scaffold
Fixation of sample (Ethanol/Methanol/Acetone/Paraformaldehyde/Other/Duration etc.) Methanol 10 min
Antigen retrieval (Enzymatic method, Heat mediated technique etc.) non needed
Permeabilization step tween 20 0.05% in PBS
Blocking conditions (Buffer/time period, Blocking agent etc.) super block-scytek 8 min
Primary Antibody (Diluent/Dilution/Incubation time, Wash step) ab47003 1:200,24590 1:100 in pbs+1%BSA for 1 hr RT,wash in tween 20 0.05% in PBS 3 times for 5 min while shaking
Secondary Antibody (Manufacturer/Species/Diluent/Dilution/Incubation time, Wash step)
goat anti-rabbit Alexa Fluor 488 (Jackson Immuno Research Laboratories, Inc.), Goat Anti-Mouse Alexa Fluor 594 (Jackson Immuno Research Laboratories, Inc.), both 1:500
Detection method confocal
Positive and negative controls used (please specify) non, but secondary antibodies were in exact prior use with great specificity, both abcam antibodies were stained not together to generate good results
Optimization attempts (problem solving):none
How many times have you tried the IHC? twice
Have you run a "No Primary" control?
secondary antibodies were in exact prior use with great specificity
Do you obtain the same results every time? some pictures you don’t see much cross reactivity, some you see

Answer

Thank you for contacting us.
I appreciate the time taken to submit protocol information. The details you have kindly provided will provide us with vital information for our monitoring of product quality. I would also appreciate if you could please provide the order and batch details.
Reviewing this case, I would like to offer some suggestions to help optimise the results from these two antibodies in ICC/IF:
Cell fixation should be done under cold conditions. Using ice cold methanol, and optimizing it with other fixatives, such as 3-4% paraformaldehyde in PBS pH 7.4 for 15 min at room temperature.
For antibody ab24590 permeabilization is not required as the target protein is expressed in the membrane. For ab47003 I would recommend increasing the permeabilization conditions, by using more concentrated Tween 20 or 0.25% Triton X-100.
The unspecific signal obtained may be due to unspecific binding of the antibody as there has not been any blocking of unspecific binding sites. This could be improved by using 3%BSA for 30 min / 1 hour at room temperature.
Also, in order to facilitate the antibody to reach the target, incubation overnight at 4C normally gives better results than 1 hour incubation.
I hope this information is helpful. Should the suggestions not improve the results, please do not hesitate to contact me again and I will try to provide further help.

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