Goat polyclonal Secondary Antibody to Rabbit IgG - H&L (Biotin) (ab6720)

Good afternoon,

Thank you for the rapid answer to my problem.

 I would be pleased if Abcam could send me the free of charge replacement of the same Goat Polyclonal Secondary antibody to Rabbit IgG - H&L (Biotin) (ab6720).

The product must be sent to the following address:

ANSWER:

Thank you for confirming these details and for your cooperation. The details provided enable us to closely monitor the quality of our products.

I am sorry this product did not perform as stated on the datasheet and for the inconvenience this has caused. As requested, I have issued a free of charge replacement with the order number

To check the status of the order please contact our Customer Service team and reference this number.

Please note that this free of charge replacement vial is also covered by our Abpromise guarantee. Should you still be experiencing difficulties, or if you have any further questions, please do not hesitate to let us know.

I wish you the best of luck with your research.

I'm working in rat cardiac tissue fixed in 4% paraformaldehyde and embebed in paraffin.

I'm performing antigen retrieval Heat-induced with 10mM Sodium Citrate Buffer. Using normal goat serum 5% in TBST as blocking solution.Endogenous peroxidases are blocked with 3% Hydrogen Peroxide and Endogenous biotins are blocked with Endogenous avidin + biotin blocking system (ab3387)

Secondary antibody: Goat Polyclonal Secondary antibody to Rabbit IgG - H&L (Biotin) (ab6720)

Concentration or dilution 1/250

Diluent buffer 1xTBST

Incubation time 2h

Detection method: Streptavidin Protein (HRP) (ab7403)

DAB substrate kit (ab94665)

all washes were performed with TBST between every step.

This was the protocol that was used every time and have always worked until now.In The last few experimental protocols didn't work and the negative controled was stained.

When i replace this secondary antibody by other with the same caractheristics but from another brand the experimental protocol worked well again.

Attached sending two photographic records:

Negative control (Abcam) - the negative control stained (section without incubation of primary antibody and with incubation of abcam's secondary antibody) Negative control (other brand) - normal negative control (section without incubation of primary antibody and with incubation of secondary antibody from another brand)

ANSWER:

Thank you for your message and for providing this further information.

Thank you for taking the time to provide the additional details to me. These have been of great help in understanding the issue. I am sorry to hear that this product had performed unsatisfactorily for you. I appreciate the time you have spent on these experiments and would be pleased to arrange a free of charge replacement or a refund/credit note in compensation.

I look forward to hearing from you with details of how you would like to proceed.

I'm currently using Abcam products to perform imunohistochemistry on paraffin on my research protocols and I have a problem that I would like Abcam to help me.

I have been using Goat Polyclonal Secondary antibody to Rabbit IgG - H&L (Biotin) (ab6720) in my work and in my last protocols the secondary antibody didin't work well. This happened without me doing any changes in neither the protocol or the antibody.  

I've had good results with previous experiments using this same antibody and suddently I observed a very intense staining in the negative controls (where the I omitted the primary antibody).

I concluded that the problem was the secondary antibody when without doing any changes in the experimental protocol I decided to change the sencondary antibody i've been using and replace it for another sencondary antibody with the same characteristics but from another brand. With this experiement I had good results and a good negative control without staining. This showed me that the problem was that my secondary antibody (ab6720) isn't working properly.

I would like to ask what Abcam can do to help solve my problem?

ANSWER:

Thank you for contacting us.

I am sorry to hear of the difficulties that you have been experiencing using this product and wish to thank you for performing appropriate steps to determine whether the issue is with the product or protocol.

Would you mind sending me the details of your protocol including the tissue type you are working with and your sample preparations? Any images that you may have and any steps that you have changed while troubleshooting would be extremely helpful as well.
These details will not only help me to better understand your difficulties, they are also used for internal testing of the products if deemed necessary.

Would you also be able to send me the order number for this product?

The antibody is covered under our Abpromise for six months and is guaranteed to work in IHC-P. If we cannot resolve the issue you are having with the antibody then I would be happy to either send a replacement antibody or to process a refund.

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

Good afternoon, Thank you for your fast response to my e-mail. First aswering to the last two questions: I've been using Streptavidin protein (HRP) (ab7403) as for thedilution of the secondary antibody is 1/250 Nowwith regard to the form sent: 1) Abcam product code ab469 3) Description of the problem: Need more intense staining 4) Sample preparation: Species: Rat Type of sample:PFA/formalin fixed paraffin embedded sections Negative control: Removal of primary antibody 5) Fixation step Yes If yes: Fixative agent and concentration:paraformaldehyde 4% Fixation time: at least 48 hours Fixation temperature 4ºC 6) Antigen retrieval method: Heat-induced with 10mM Sodium Citrate Buffer 7) Permeabilization method: Not done 8) Blocking agent (eg BSA, serum…):Normal Goat serum Concentration 5% in TBST Blocking time: 1h Blocking temperature: room temperature 9) Endogenous peroxidases blocked? Yes (3% Hydrogen Peroxide) Endogenous biotins blocked? Yes (Endogenous avidin + biotin blocking system ab3387) 10) Primary antibody (If more than one was used, describe in “additional notes”) : Anti-Survivin (ab469) Concentration or dilution 1/500 Diluent buffer1xTBST Incubation time Overnight 11) Secondary antibody: Goat Polyclonal Secondary antibody to Rabbit IgG - H&L (Biotin) (ab6720) Concentration or dilution 1/250 Diluent buffer1xTBST Incubation time 2h 12) Washing after primary and secondary antibodies: Yes Buffer: 1x TBST Number of washes at least 3 times 13) Detection method: Streptavidin Protein (HRP) (ab7403) DAB substrate kit (ab94665) 14) How many times have you run this staining? Several Times Do you obtain the same results every time?yes almost every time What steps have you altered to try and optimize the use of this antibody? Altering the dilution of primary antibody, altering the time of incubation of the primary antibody, altering the time of incubation of the cromogen. Best regards,

ANSWER:

Thank you for providing that extra information. It has helped greatly to understand what you have been doing and what may help to improve the signal of the staining. There are a few areas that I would suggest would be worth optimising (if you have not already done so) which may lead to an improved signal. 1. Fixation You have stated that you are fixing the tissue for a minimum of 48 hours. The ideal fixation time will depend on the size of the tissue block and type of tissue but usually 18-24 hours seems ideal for most applications. Over-fixation can lead to the epitope being masked. 2. Antigen retrieval Antigen retrieval can be used to overcome the masking mentioned for the fixation. Depending on the level of masking, different conditions of antigen retrieval may be required. For example, if using a microwave we would usually suggest trying retrieval for 5, 10, 15 and 20 minutes to see which produces the optimal results. 3. Hydrogen peroxide blocking I would suggest performing the hydrogen peroxide blocking following the incubation of the primary antibody if you have not already tried this. The hydrogen peroxide can sometimes affect sensitive epitopes and by performing the step following the incubation with the primary antibody this will not affect the staining. 4. Primary antibody incubation You mention that you have optimised this step by changing the dilution and incubation time. If you have not already done so I would suggest attempting the incubation for 1-1.5 hours at room temperature with agitation. 5. Detection method Different techniques can be used to directly amplify the signal. Currently you are using streptavidin-HRP conjugate which is labelled in a 1:1 ratio. It is possible to employ avidin which has been labelled in a 3:1 ratio with HRP using kits such as Piercenet product 32020. Although avidin can cause greater background compared to using streptavidin. Alternatively HRP polymer can be employed. This consists of using a secondary antibody which is attached to a polymer-HRP complex. Abcam has the following product to offer, ab2891, however this is a little pricey. I would suggest trying to optimise the steps 1-4 and if sufficient progress is not made contemplate employing one of signal amplification methods mentioned. We have quite a detailed guide to IHC which may be of help to you. This outlines many of the different experimental parameters which need to be considered when performing IHC and suggestions to improve experiments: http://www.abcam.com/ps/pdf/protocols/ihc_p.pdf I hope this information has been of help and your staining improves. If you want any further information or help please do not hesitate to contact us again.

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.