Anti-TLR9 antibody (ab13928)

DESCRIPTION OF THE PROBLEM No signal

SAMPLE Whole spleen cells from H2d mice

PRIMARY ANTIBODY Anti TLR9 (rabbit polyclonal to TLR9) ab13928, 0.5 mg/ml tested at 3 different concentrations: 0.5?g, 2.5?g and 5?g for 10.6 cells incubation: 30 minutes 2 washes PBS/saponin buffer (1600rpm,7min,4?C)

SECONDARY ANTIBODY ZYMED FITC-Goat anti rabbit IgG (H+L) dilution 1/250, 1/500 and 1/1000 incubation: 30 minutes 2 washes PBS/saponin buffer (1600rpm,7min,4?C)

DETECTION METHOD FACScalibur flow cytometer using CellQuest software

POSITIVE AND NEGATIVE CONTROLS USED FITC CD8 staining no other controls

ANTIBODY STORAGE CONDITIONS 4?C, liquid

NUMBER OF CELLS USED 10.6 cells/well

PERMEABILIZATION STEP permeabilization: 10 minutes incubation with PFA/saponin solution

2 washes PBS/saponin buffer (1600rpm,7min,4?C)

HOW MANY TIMES HAVE YOU TRIED THE APPLICATION? 2 HAVE YOU RUN A "NO PRIMARY" CONTROL? Yes DO YOU OBTAIN THE SAME RESULTS EVERY TIME? Yes

WHAT STEPS HAVE YOU ALTERED? adding different concentrations of both primary and secondary antibody

ADDITIONAL NOTES Was the anti TLR9 antibody tested on whole spleen cells? I had no signal at all using the protocol described above: Would it be more efficient on purified dendriticic cells?

ANSWER:

Thank you for your enquiry and for submitting the completed questionnaire on-line. We are very sorry to hear that you are having problem with this antibody.

This product has been characterized on Raw cells and on thioglycolate-elicited mouse macrophage cells. Unfortunately, we have not tested it on mouse spleen cells so we do not have any results which we could share with you. It may be worth running an experiment with mouse macrophages as positive control to see if the signal is getting stronger. How do you isolate the cells from the spleen? Do you digest the tissue? Are the cells intact after the pretreatment? Have you tested if they are not damaged?

The protocol seems to be absolutely fine, although you have not mentioned the concentration of the saponin used to permeabilize the cells. We usually recommend 0.1-0.5% saponin for FACS analysis.

The other thing which may cause the problem is that the antibody may have gone off. As we understand from your e-mail, you stored the antibody at 4oC. How long have you stored the antibody in the fridge. We would recommend keeping this particular antibody (as the datasheet states) at 4oC for only a short period of time (max for one week). For long term it should be stored at -20 or -80 oC.

Have you tested the secondary antibody with another primary antibody? Does it work properly?

We hope this will help. Should you still have problem with this antibody, then please do not hesitate to contact us again.

Customer's question: I am inquiring about your anti-TLR9 antibody. I would like to use it to detect TLR9 in RAW cells by flow cytometry. The product sheet references these cells in the application notes but does not say if the staining was intra-cellular or extra-cellular. To obtain results like the histogram shown on the product sheet, was the staining intra-cellular or extra-cellular? How were the cells processed before staining?(EDTA, scraping, or stained before removal from flask?) Was anything used to block non-specific binding?

Thank you for your help!

ANSWER:

There was intracellular staining carried out. Cells were fixed with Cytofix from BD. Please let me know if you need any other information.