Anti-Von Willebrand Factor antibody (ab6994)

Unfortunately we are still struggling with our use of anti-VWF and anti-Fox-3. At one point we thought Fox-3 was working but we didn't get consistent staining.

As suggested we implemented the trypsin enzymatic antigen retrieval system into our protocol. We did it from 10-35 minutes (multiple samples at multiple time points) at room temperature and at 37 degrees as suggested.

We did titrations of our primarys and secondaries with multiple samples. For anti-VWF we used 1:200-1:800 for the primary (about 60 samples were done trying this) and Fox 3 (60 samples) at 1:200-1:1000. Nothing has been demonstrated to work effectively except an anti-BrdU antibody we purchased from another company and used on separate samples from the same animals. With our secondaries, as advised, we took drops of secondary directly and put them on a slide examining them. The drops did fluoresce suggesting that they were not burned out. Also, we did secondary titrations varying from as low as 1:50 as recommended to 1:200. Nothing provides reliable staining.

We don't know how to proceed at this point. What would you suggest? Are there other antibodies you would suggest?

ANSWER:

Thank you for your email.
I am so sorry to hear that both antibodies are not working as expected.

At this point - after having tried so many different protocol options - I would suggest that the primary antibody is not working as it should. Please let me know your order or PO number for both antibodies.

I'd be happy to replace both of them.
For VWF, we only have ab8822 that has been tested in IHC-Fr and rat, and it is FITC conjugated. We have other antibodies for VWF that are tested in IHC-Fr but not on rat: e.g. ab11713, ab9378 or ab778.

For Fox3, we have only one other antibody: ab104224 which has not yet been tested in IHC-Fr , but was tested on rat samples. If you like to try this one on frozen sections, I would give you in addition also a testing discount code.
This would also apply for the VWF antibodies that are not tested yet on rat.

Here is some more information in this regard:
For UNTESTED species and/or applications, we have established a testing discount program. Here is a brief description of how it works:
The testing discount program is for customers who like to use an antibody/protein/kit on an untested species/application. You would purchase the antibody/protein/kit at full price, test it and submit an Abreview with your data (positive or negative). On your next order you will receive a discount for ONE antibody/protein at the full price (100%) of the antibody/protein you have tested, or a full price discount for the amount paid for the kit. The terms and conditions applicable to this offer can be found here: http://www.abcam.com/collaborationdiscount.
This programapplies to ELISA kits and other kits we offer.

Alternatively, a credit or refund can be arranged.
Please let me know which options are the best for you and I'd be happy to arrange them for you.

I look forward to hear back and resolve this problem for you.

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1) Antibody is stored at -4 until use, just a normal freezer. When I use it I keep it in a refridgerator for no longer than 2 days.

2) Could you clarify this point? Are you saying I should not have added the PBS to the antibody vial? Only 50ul was shipped--essentially I could see no ab in the vial. I had to add some PBS to the vial because when I recieved it there was nothing to pipette out. When you say "increase antibody concentration" do you mean "decrease antibody concentration?." Also--I did not want to dilute the antibody before doing my titration experiments because i was nervous to over dilute rendering the rest of the ab useless.

3) I will use this temperature.

4) I just dip them in a copland jar with PBS for 2 minutes per jar so a total of 4 minutes. This was recommended in another company's antibody's primary antibody application videos for ab staining.


--Over the weekend I extended the period of antigen retrieval to 25 minutes, increased the conc of secondary to 1:50 and increased the conc of primary fox 3 to 1:500. I was able to get Fox 3 to work for the most part. VWF still doesn't display any fluorescence. What should I try now? I will read the paper.

ANSWER:

Thank you for your email.

To answer your questions and make some comments/suggestions that hopefully will help you further:

1) The antibodies (shipped as is) should be stored aliquoted and stored at -20 or -80 degree. This would also apply to the 1/10 stock solution as well. Please make sure to avoid freeze-thaw cycles. For ab6994, a brief period of storage at +4 degree for 1-2 weeks would be fine, but for long term storage -20 or -80 degree is recommended.

2) For ab6994, you should have received 100 ul, and for ab104225 you should have received 50 ul. You can spin the vials briefly before opening and the liquid should be at the bottom of the tube and it would be visible. In thecase of these 2 antibodies, diluting them to 1/10it might be OK as they are either whole antiserum (contain other proteins) or contain lots of BSA as stabilizer.
In general, it is recommended to dilute the antibody to the working solution just before use.

A 1/100dilution would be an increase in antibody concentration compared to 1/500 (which is more dilute), as 1/100 contains more antibody than 1/500.

3) Yes, the higher temperaturewill help with the antigen retrieval.

4) Yes, this is probably fine. But in case of no signal, you might consider reducing the time to e.g. 2x 30 sec.

I'm glad to hear you are getting now signal with the Fox 3 antibody. As for the VWF antibody, would also use more primary and secondary antibody, increase the retrieval temp to 37 degrees and reduce the wash time. Yes, checking the reference could provide additional helpful information.

I wish you good luck and look forward to hear back.

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The trypsin antigen retrieval did not work. I'd like to review what I did to see if there are any large errors.

Rats wre perfused in situ and brains were preserved in 4% paraformaldehyde solution overnight. We purchased the paraformaldehyde commercially pre-prepared. We then sucrose protected the brains (after speaking with an abcam rep) in a sucrose series to freeze protect them. We then froze the brains for a period of about a month.

I recieved the Fox-3 antibody from abcam as a 50ul concentrate. In reading the antibody reviews online I saw that our target antibody concentration would be between 1:500-1:2000. So I initially added 500ul to the 50 ul concentrate to have an initial 1:10 dilution (roughly). Then I took 1 ul of this working solution and diluted it with 200 ul PBS to get my 1:2000 dilution. I followed this with 1ul working solution in 100 ul PBS to get my 1:1000 dilution. Then, 2:100ul PBS to get my 1:500 dilution. I did this because an abcam representative said I should shoot for 100ul per slice.

I took my test slices, washed them in PBS for 5 minutes, then outlined them in a histology pen, and applied the primary. I spoke with an abcam rep last week who advised us not to block unless we had too much non-specific staining. I used the trypsin enzymatic retrieval kit you recommended which entained mixing the trypsin concentrate with a diluent included in the kit at a ratio 1:1. I applied 100ul of this per slice for 15 minutes at room temperature under a fume hood (not in the dark--this was not required in the literature). I then washed twice with PBS, and I applied the primary on a counter top at roomtemperatureand then stored in at 4 degrees c overnight in a dark container. After this, I rinsed the slices in PBS, and thenbathed the slices twice in PBS to get the primary off, and then applied the Dylight secondary (dilution info below) for one hour at room temp in a dark environment. I then washed once with PBS and applied the DAPI mounting medium we purchased from abcam. I coverslipped the slides and left them for about an hour at 4 degrees c in a dark slide holder. I then attempted microscopy.

Secondary info: (Donkey anti rabit Dylight 488) from its stock solution and read online that a 1:50-1:200 dilution would be optimal so I tried 1:100 which should have produced at least some fluorescence. I took the equivalent of 1ul Dylight to 99ul PBS done in the dark..

The VWF antibody was prepared the exact same way except the suggested concentrations were slightly different.
I took the 100ul concentrated antibody that abcam shipped and immediately added 900ul for a 1:10 working solution dilution. Then I did an experiment with 1:200 (5ul working solution in 95ul pbs); 1:500 (2ul working in 98 ul pbs); and 1:800 (2 ul working in 198 pbs). Secondary was applied at same conc.

Where should we go from here?

ANSWER:

Thank you for your email.
I am sorry to hear the antigen retrieval step did not improve your results.

I have looked throught your protocol you sent me and have the following suggestions, comments and questions:

1) You are making a 1/10 stock solution for each antibody. How do you store the antibody afterwards?

2) Also, in my understanding, diluting the entire vial of antibody - although it makes it easier for pipetting - can lead to a loss of antibody to the tube wall due to absorption andan increase in volume. Thus, increasing the primary antibody concentration might be necessary(e.g. 1/100).

3) For the enzymatic antigen retrieval, a temperatue of 37 degree would be recommendedas this is the temparature where the enzyme is most active.

Here is the link to our antigen retrieval protocol page: http://www.abcam.com/index.html?pageconfig=resource&rid=11488. In section 3, you can find information about the enzymatic retrieval.

4) In your protocol description, you mentioned: "I rinsed the slices in PBS, and then bathed the slices twice in PBS to get the primary off". How long did you bathe the slides in PBS each time? If this is done for too long, you might potentially also lose primary antibody.

5) As for the secondary antibody, it will only bind specifically if the primary antibody is present. Thus, doing the antigen retrieval at a higher temperature and using potentially more primary antibody should help with that. In addition, you might also want to try a lower dilution and longer incubation time with the seconadry antibody (e.g. 1/50 for 2 h).

6) You are using perfused frozen sections which we abbreviate IHC-FoFr.
For the VWF antibody (ab6994) this application has been tested, and it was added based on the following publication:
Fujimoto KL et al. Naive rat amnion-derived cell transplantation improved left ventricular function and reduced myocardial scar of postinfarcted heart. Cell Transplant 18:477-86 (2009). IHC-FoFr; Rat. PubMed: 19622235
It might be worthwhile checking this publication for additional protocol details or contacting the authors for the details of their technique.
In addition, a customer submittedthe following Abreview for IHC-FoFr:
http://www.abcam.com/index.html?datasheet=6994&tab=abreviews&intabreviewid=11467

As for the FOX3 antibody (ab104225) this application has not been tested yet, and we do not have information if and how well IHC-FoFr would work with this antibody. Thus, we also do not havea specific protocolestablished for this antibody. Since IHC-P was tested with ab104225, IHC-FoFr might work as well. Here is the link to the Abreview a customer submitted for IHC-P:
http://www.abcam.com/index.html?datasheet=104225&tab=abreviews&intabreviewid=25847

I have also attached a paper describing how heat-inducedantigen retrieval can be used on frozen sections.

I hope this information will be of help to you. Please let me knowif these suggestions are improving your results or not.

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Thank you for the help today. I have already purchased ab970 (trypsin enzymatic antigen retrieval) after our conversation and will let you know how that works. I took 5 ul of secondary and it did flouresce so the antibody is not dead. I will try antigen retrieval when it arrives.

Again, here are the ab's that I purchased.

Primaries
ab6994
ab104225

Secondary
ab96891

I had purchased them after speaking with a representative at tech support at abcam, but you are very knowledgeable--do you know of a secondary that maybe more appropriate for the two primaries? We will purchase something new if it is more likely to work.

ANSWER:

Thank you for your reply.
I'm glad to hear that the secondary antibody shows fluorescence.

As for selecting another secondary antibody, could you please let me know which fluorophore or wavelength range is covered by your miscroscope? Based on that informationI can then select a different antibody for you.

Please let me know. Thank you!

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IHC-Fr with adult rat brain (hippocampus)
no signal at all
20 um sections
fixation: PFA 4% overnight
Ab: ab6994: 1/200, 1/500, 1/800 o/n 4 degree
ab104225: 1/500, 1/1000, 1/2000 o/n 4 degree
block: none
secondary: 1/200

suggestions: try AR (enzymatic), check if secondary works (fluorescent plate reader or fluorescence micrscope, with other primary Ab)

ANSWER:

Thank you for contacting us.
I am sorry to hear that the antibodie might not be working as expected.

As we discussed over the phone the following troubleshooting tips might help in finding the cause of the problem:

1) Try antigen retrieval (enzymatic) using one of the products we discussed (ab64201, ab64220, ab64205 or ab970).

2) Check if secondary antibody works (e.g. with fluorescent plate reader or fluorescence micrscope, or with another primary Ab)

Please let me know if and how your results improving.
I wish you good luck and look forward to hear back from you.

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