Anti-beta Galactosidase antibody (FITC) (ab6641)

No staining in mouse tissue in IHC-Fr.

ANSWER:

Thank you for calling Abcam earlier today.

I am sorry about the problems you were having with ab6641 in your staining. As we talked about on the phone today, I am sending you ab12081 as a free of charge replacement and ab6881 aswell. Your new order/reference number is ******.

If there is anything else I can help you with, please let me know.

BATCH NUMBER -- NOT SPECIFIED -- ORDER NUMBER 148729

DESCRIPTION OF THE PROBLEM I have regularly used your ab6641 to detect beta-gal labeled human cells injected into mouse hearts. It has worked beautifully in the past. My most recent (purchased 5/19/06) vial is not performing well. I have verified the presence of cells using an x-gal stain and an antibody that recognizes human nuclei. I was hoping you could send me a new vial of the ab6641, preferably a new batch, so that I could try this antibody again.

SAMPLE beta-gal labeled human cells in mouse hearts

PRIMARY ANTIBODY ab6641

DETECTION METHOD microscopy

ANTIBODY STORAGE CONDITIONS 4 degrees for 1 week, -20 degrees in aliquots

FIXATION OF SAMPLE paraformaldehyde

ANTIGEN RETRIEVAL none

PERMEABILIZATION STEP 0.1% Triton 10 min

BLOCKING CONDITIONS 10% serum with 1% BSA in TBS 1 hr

HOW MANY TIMES HAVE YOU TRIED THE APPLICATION? 50 HAVE YOU RUN A "NO PRIMARY" CONTROL? Yes DO YOU OBTAIN THE SAME RESULTS EVERY TIME? Yes

WHAT STEPS HAVE YOU ALTERED? none since the antibody performed well

ANSWER:

Thank you for your enquiry.

I am sorry to hear that you have been having difficulties with this antibody. Please can you provide me with details of the lot numbers that you have been using; both the successful lot in addition to the lot that you have not been able to obtain satisfactory results with so that I can try and trace any problems that we may have experienced with this lot.

I appreciate your patience in this matter.

How was this antibody tested? What cell line was used?

ANSWER:

Thank you for your enquiry. This antibody was tested in IEP (Immunoelectrophoresis), showed a positive and a single arc at the right position against anti-FITC, anti-Rabbit IgG, and b-Gal. The D/P ratio has to be in the optimal range of 2-5.

Please contact us again if you have any additional questions.

Thanks. Answers are: 1. We tried at the ab alone for immunofluorescence at 1:1000. We will repeat today at 1:200. 2. Ab dilution buffer is TBS+0.5%BSA 3. Wash buffer is the same.

ANSWER:

Thank you for your rapid reply and this information.

I would recommend to use PBS containing tritonx100 (0.3%) to dilute the antibody and to incubate the sections in this buffer containing 10% normal serum for 1 hour prior to adding the primary antibody. The presence of triton is very important to permeabilise the cells and help the antibody penetrate. Please use PBS to wash the sections prior to the blocking stage and after the antibody incubation stage. You have not mentioned how long you have tried incubating the antibody for, can I please suggest trying a range of incubation times too?

I look forward to hearing how you get on with those modifications,

DESCRIPTION OF THE PROBLEM No staining at 1:500-1:5000, with high background coming up at 1:500. We used this ab on frozen, acetone-fixed sections for both immunohistochemistry (ab6641 followed by an anti-fitc alk.phos. or anti-rabbit-HRP) and immunofluorescence. The secondary abs are good ab that we frequently use and that we know give low background.

SAMPLE mouse lymph node from a mouse expressing beta-gal in specific cell types. beta gal expression is easily detected by x-gal development.

POSITIVE AND NEGATIVE CONTROLS USED There was no positive control for the primary, except that we know this tissue expresses lacz by xgal staining. The negative control was mouse tissue from a mouse not expressing lacz. Background was there in the neg control, but it would have been workable if there was an obviously detectable signal on our lacz+ tissue.

ANTIBODY STORAGE CONDITIONS At 4 degrees

FIXATION OF SAMPLE Tissue frozen in OCT, cut at 7 um, dried for 1 hour, acetone fixed cold for 10 min, and stained. We do quite a bit of IHC/IF and this procedure works well for our staining with other ab.

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

ADDITIONAL NOTES In your testing of this ab for frozen tissue, did you use acetone fixation?

ANSWER:

I'm sorry to hear you are experiencing problems with ab6641. This antibody is the FITC conjugated version of ab616, one of our most popular antibodies. It has been tested on frozen sections fixed with cold methanol or paraformaldehyde.

In order to help you I would appreciate if you could clarify: 1) if you have used the antibody alone or always with secondary antibodies 2)what incubation time you have tried 3) what antibody dilution buffer you have used 4) what blocking buffer you have used

If you can provide the lot number of this vial I can check if we have received any complaints and if you can provide your order number I can also look at possible shipping delays which would suggested the vial was damaged during transport.

I'm sorry for the delay in resolving this matter and appreciate if you can provide those few details, I look forward to hearing from you,