Anti-CD62E antibody (ab18981)

1. Deparaffinize/hydrate sections:
a) Incubate sections in three washes of xylene for 5 minutes each.
b) Incubate sections in two washes of 100% ethanol for 10 minutes each.
c) Incubate sections in two washes of 95% ethanol for 10 minutes each.
2. Wash sections twice in ddH2O for 5 minutes each.
3. Wash sections in PBS for 5 minutes.
4. For antigen unmasking, heat sections in 10 mM sodium citrate buffer (pH 6.0) for 1 minute at full power followed by 9 minutes at medium power. (Keep slides fully immersed in buffer and maintain temperature at or just below boiling.) Cool slides for 20 minutes after antigen unmasking.
5. Wash sections in ddH2O three times for 5 minutes each.
6. Incubate sections in 1% hydrogen peroxide for 10 minutes.
7. Wash sections in ddH2O three times for 5 minutes each.
8. Wash section in PBS for 5 minutes.
9. Block each section with 100-400 µl blocking solution for 1 hour at room temperature.
10. Remove blocking solution and add 100-400 µl diluted primary antibody to each section. (Dilute antibody in blocking solution.) Incubate overnight at 4oC.
11. Remove antibody solution and wash sections in PBS three times for 5 minutes each.
12. Add 100-400 µl secondary antibody, diluted in blocking solution, to each section. Incubate 30 minutes at room temperature.
13. If using ABC biotin/avidin method, make ABC reagent according to the manufacturer’s instructions and incubate solution for 30 minutes at room temperature.
14. Remove secondary antibody solution and wash sections three times with PBS for 5 minutes each.
15. Add 100-400 µl ABC reagent to each section and incubate for 30 minutes at room temperature.
16. Remove ABC reagent and wash sections three times in PBS for 5 minutes each.
17. Add 100-400 µl DAB reagent to each section and monitor staining closely.
18. As soon as the section turns brown, immerse slides in ddH2O.
19. If desired, counterstain sections in hematotoxylin for 10 seconds.
20. Wash sections in ddH2O two times for 5 minutes each.
21. Dehydrate sections:
a) Incubate sections in 95% ethanol two times for 10 seconds each.
b) Repeat in 100% ethanol, incubating sections two times for 10 seconds each.
c) Repeat in xylene, incubating sections two times for 10 seconds each.
22. Mount coverslips.

Thank you for your enquiry.

I can confirm that ab18414 can be stored at 4oC as described on the datasheet. This should not be aliquotted and frozen.

The others I would recommend to aliquot and freeze immediately. Keeping them at 4oC for a few weeks should not significantly affect the staining. However, we would recommend to follow the individual storage instructions provided on the individual datasheets as soon as possible after delivery. This will help to ensure that the antibody remains stable and that we can continue to provide our guarantee.

I hope this will be helpful to you. If you have any further questions, please do not hesitate to contact us.

I am very pleased to hear you would like to accept our offer and test ab54426 in IHC-P.

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This code will give you: 1 free primary antibody before the expiration date. To redeem this offer, please submit an Abreview for ab54426 used in IHC-P and include this code in the “Additional Comments” section so we know the Abreview is for this promotion. Please remember that submission of the Abreview is sufficient for the discount code to become active.

For more information on how to submit an Abreview, please visit the site: www.abcam.com/Abreviews.

Remember, we publish both positive and negative Abreviews on our datasheets so please submit the results of your tests. The code will be active once the Abreview has been submitted and can be redeemed in one of the following ways:

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With regards to the isotype controls:
For ab2213, the light chain type is unknown, so I would recommend to use a general IgG2a isotype control rather than a specific IgG kappa chain isotype control. For example the following product:

ab91361 Mouse IgG2a [ICIGG2A] - Isotype Control
https://www.abcam.com/index.html?datasheet=91361 https://www.abcam.com/index.html?datasheet=91361.

However, please note this is not tested in IHC-P. Unfortunately, I am sorry we do not have any suitable isotype controls tested in IHC-P in this case.

ab27478 is a suitable isotype control for ab18981
ab91353 is a suitable isotype control for ab54426

Any feedback that you can provide regarding ab54426 will be greatly appreciated, whether positive or negative. If you have any further questions, please do not hesitate to contact us. We look forward to receiving your Abreview and wish you luck with your research.

The terms and conditions applicable to this offer can be found here: www.abcam.com/collaborationdiscount.

Thank you for contacting us.
I am sorry to hear that the antibody does not seem to work as expected.

As we had discussed, I would suggest to try the following tips to reduce the background as well as shift the MW to a higher band:

1) use less primary and/or secondary antibody
2) add fresh reducing agent to the loading buffer (e.g. DTT or beta mercaptoethanol)

Please let me know if these suggestions are of help or not. If not, we can discuss other options to resolve the problem.

I look forward to hear back from you.

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I agree with Heather that it would be useful to have a negative control sample. I may be mistaken but if E-selectin is only expressed exclusively in endothelial cells, perhaps using an extract from a different cell type as a negative control will be helpful. Alternatively, it may be helpful to look at the literature where E-selectin is blotted for to see what controls others have used.

Also I am happy to look at your western blot if you'd like to send it to me as an attachment.

I hope this is helpful. Please contact me again if you have any further questions.

Thank you for your phone call. I just wanted to follow up with you and send you the suggestions we had discussed over the phone: 1) use peptide in 10-fold excess 2) incubate peptide with Ab for 1-2 h at RT before using for WB 3) avoid preparing an "antibody only"-control simultaneously with the peptide-antibody, just use fresh antibody for doing the WB (due to potential absorption of the antibody to the tube wall which will lead to a lower antibody concentration in this tube). I hope this information is helpful to you. Please let me know how everything works out. I wish you good luck. Please do not hesitate to contact us if you need any more advice or information.