Endogenous Avidin + Biotin Blocking System (ab3387)

The attached MSDS does not include the 1st page.  Please send me a complete MSDS for product number ab3387.  

ANSWER:

Thank you for contacting us regarding this matter.

We have updated the MSDS and have fixed this issue. We would like to thank you for bringing this to our attention. I gave included the updated MSDS here but it is available on the webpage for this product as well.

I hope this information is helpful to you. Please do not hesitate to contact us if you need any more advice or information.

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Thanks for the reply.

We never did in our laboratory this protocol with Egg white and milk. Could you explain in more detail how and in what part of the procedure should I enter them.

I'm very pleased with the attention and speed that Abcam that serves customers.

Best regards,

ANSWER:

Thank you for your inquiry. My colleague is not in the office today and I am happy to answer your question instead of her. Please find below a protocol for blocking biotin with egg white:
1- Wash twice in TBS 0.05M pH7.5, to which 0.01% Tween 20 has been added (TBS-T).
2- Briefly blot the slides without letting them dry and then Apply egg white in PBS-BSA-NaN3 as a blocking agent (one egg white in 100 ml PBS + NaN3) (Miller RT et al, Appl Immunohistochemistry 5(1): 63-66, 1997). Incubate for 10 min. minimum. 3- Wash once in water.
4- Incubate with skim milk 5% in TBS-T for 10-30 min. (Miller RT et al, Appl. Immunohist & Molecular Morphology, 71(1): 63-65, 1999)
NOTE: skim milk may contain weak proteases which will be useful to further unmask antigens, but may reduce immunostaining by acting on primary Abs.
5- Wash once in TBS-T (residual milk can contribute to blocking).
This biotin blocking step can be added to the IHC protocol anywhere between the antigen retrieval and the immunostaining. I suggest to add this step to the general blocking step with serum.
Since you do not have any experience with egg white as blocking agent, I can recommend to use our biotin blocking kit ab3387:
http://www.abcam.com/index.html?datasheet=3387 http://www.abcam.com/index.html?datasheet=3387.
This will be easier to use and will also require less optimisation.
I hope this information was helpful and wish you good luck with your experiments.

I made more tests and still gives unexpected results, osteoblasts were labeled and not all osteoclasts were labeled.

I tried two new forms of antigen retrieval:

1- used skimmed milk to block the endogenous biotin

2- used bovine serum albun (BSA) 20% (the twice of the original protocol)

I'm sending in attached the pictures of my tests to you analyse my mistake (if possible).



Responding you questions...

1- I decalcificate my samples.

2- I tried use milk to block the endogenous biotin, but if the osteoblasts labeled for endogenous biotine the negative control would not going label too?

3- I did antigen retrieval using 10 mM citrate buffer (pH 6.0) in microwave



I don't know what else to try, because I´ve used almost half of antibody in test and still didn't work.



Looking forward to your answer.

ANSWER:

Thank you for contacting us.
Please be aware that the biotin problem is still not solved by using milk or BSA: those two will block the non-specific protein binding sites, but not the endogenous biotin. You can block the endogenous biotin by using either a ready-to-use avidin solution like our ab3387:
http://www.abcam.com/index.html?datasheet=3387 http://www.abcam.com/index.html?datasheet=3387.
Another, more simple method would be using egg white followed by skim milk. Egg white contains avidin, and skim milk is actually rich in biotin, and you need the free biotin to make the bound avidin unaccessible to your amplification system. Please rinse well after this step.
Regarding the antigen retrieval, I fear 10 min are not enough to crack the methylen bridges formed by the fixation agent. Please increase the time to a min of 20 min. so that the antibody can bind its target.
If you will see no improvement after those tips I will gladly provide a free of charge replacement, credit note, or refund (if the problem has been reported within 6 months of purchase).
I hope this information is helpful to you. Please do not hesitate to contact us if you need any more advice or information.
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I am having trouble with Ki67 antibody (cat#ab15580). I used it in mouse tissues such as kidney, liver, testis, brain, cartilage etc. fixed in Bouin’s fixative and formaldehyde. The staining was with extremely high background. I tried 1:100 dilution at RT for 2hrs and also 1:200 O/N +4C0.

Besides the background staining, it stained cytoplasm of the cells! Here is my protocol: Retrieval: 10 mM sodium citrate buffer, pH6.0 in microwave, 5 min, twice. Cool down in the buffer for about 20 minutes. Wash in deionized water 3 times for 5 minutes each. Blocking for endogenous peroxidase with 3% H2O2 prepared in methanol for 20min, RT. Wash in deionized water 3 times for 5 minutes each. Blocking with UV block (Dako) for 7min, RT. Incubation with primary antibody Ki67 (ab15580) in antibody diluent (Thermo). Wash in PBS, 3 times, 5min each. Incubation with secondary antibody in PBS (vector, biotinylated anti rabbit; 1:500) for 30min, RT. Incubation with ABC substrate (Vector) for 30min, RT. Wash in PBS, 3 times, 5min each. DAB staining, wash, counterstaining and mounting with Cytoseal.

When I compared the Ki67 and PCNA (another proliferation marker) on the same slide, the difference was obvious. By the way, my negatives worked fine. I can send you some pictures as well. Thank you and I look forward to your reply.

ANSWER:

Thank you for contacting us.

Is the UV block from DAKO a protein block? We typically recommend performing a protein block using 10% normal serum from the host species of your secondary antibody. For example, if your secondary is goat anti-rabbit, we recommend blocking with 10% normal goat serum.

Additionally, when using ABC detection, endogenous avidin and biotin can sometimes cause diffuse, weak, cytoplasmic staining. To prevent this, you can use a system like ab3387.

I hope this will help to improve your results. If not, I will be happy to offer you a free of charge replacement, credit, or refund if you have purchased this antibody in the past six months. Please let me know your original order number and how you would like to proceed and I will be happy to assist you further.

Good morning, I'm currently using Abcam products to perform imunohistochemistry on paraffinon my research protocols and I have some doubts that I would likeAbcam tohelp me. I worked with Anti-Survivin antibody (ab469) as a primary antibody in a 1/500 dilution. I useda goat polyclonal secondary antibody (ab6720) as well as endogenous Avidin + Biotin Blocking System (ab3387) and DAB Substrate (ab94665)kit as a cromogen. With this experience Iwas able to get favorable results, however I would like to know how to get a more intense staining?I tried to optimize the dilution of primary antibody as well as the exposure time of DAB butcould not get significant changes. Could you give mean advise on how to get some more intense staining? Another question about the primary antibody Anti-Smac / Diablo (ab8114) is what dilution do you recomend using in IHC-P protocols? I read the datasheet where you say to use a concentration of 5ug/ml but I can't understand how to perform this concentration/ diluition. Best regards,

ANSWER:

Thank you for contacting us. I'm glad to hear you have been getting good staining with ab469. In order to help you obtain more intense staining would you mind filling in the form which I have attached to this email. It will allow me to more fully understand the protocol which you have been performing and any areas in which it may be worthwhile to optimise. Could you also answer the following questions: 1. which HRP conjugate have you been using (streptavidin-HRP? could you tell me the catalogue no. etc) 2. in what dilution have you been using the secondary antibody? ab8114 is supplied at a concentration of 1 mg/mL. The recommended working concentration of this antibody when using it for immunohistochemistry with paraffin embedded sections is 5 µg/ml. This equates to a dilution of 1/200. This is only a guideline and may need to be optimised for the staining which you are performing. I hope this information has been of help and look forward to your reply.