Goat Anti-Rabbit IgG H&L (Biotin) (ab6720)

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Overview

  • Product name

    Goat Anti-Rabbit IgG H&L (Biotin)
    See all IgG secondary antibodies

  • Host species

    Goat

  • Target species

    Rabbit

  • Tested applications

    Suitable for: WB, ELISA, Immunomicroscopy, Dot blot, ICC/IF, IHC-Fr, IHC-Pmore details

  • Immunogen

    Rabbit IgG whole molecule

  • Conjugation

    Biotin

Properties

  • Form

    Liquid

  • Storage instructions

    Shipped at 4°C. Store at +4°C short term (1-2 weeks). Upon delivery aliquot. Store at -20°C or -80°C. Avoid freeze / thaw cycle.

  • Storage buffer

    pH: 7.20
    Preservative: 0.01% Sodium azide
    Constituents: 0.42% Potassium phosphate, 0.87% Sodium chloride, 1% BSA
  • Concentration information loading...

  • Purity

    Affinity purified

  • Purification notes

    This product was prepared from monospecific antiserum by immunoaffinity chromatography using Rabbit IgG coupled to agarose beads followed by solid phase adsorption(s) to remove any unwanted reactivities.

  • Conjugation notes

    Biotinamidocaproate N-Hydroxysuccinimide Ester (BAC) Biotin/Protein Ratio: 10-20 BAC molecules per Goat IgG molecule

  • Clonality

    Polyclonal

  • Isotype

    IgG

  • Research areas

Applications

Our Abpromise guarantee covers the use of ab6720 in the following tested applications.

The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.

Application Abreviews Notes
WB 1/5000 - 1/20000.
ELISA 1/50000 - 1/200000.
Immunomicroscopy Use at an assay dependent concentration.
Dot blot Use at an assay dependent concentration.
ICC/IF 1/1000 - 1/5000.
IHC-Fr Use at an assay dependent concentration.
IHC-P Use at an assay dependent dilution.

Images

  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Goat Anti-Rabbit IgG H&L (Biotin) (ab6720)
    Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Goat Anti-Rabbit IgG H&L (Biotin) (ab6720)

    IHC image of beta actin staining in a section of formalin-fixed paraffin-embedded [human normal colon]*. The section was pre-treated using pressure cooker heat mediated antigen retrieval with sodium citrate buffer (pH6) for 30mins. The section was then incubated with ab8227, 1/1000 dilution, for 15 mins at room temperature. A goat anti-rabbit biotinylated secondary antibody (ab6720, 1/1000 dilution) was used to detect the primary, and visualized using an HRP conjugated ABC system. Streptavidin HRP was used, ab7403 at a 1/10000 dilution. DAB was used as the chromogen (ab103723), diluted 1/100 and incubated for 10min at room temperature. The section was then counterstained with haematoxylin and mounted with DPX. The inset negative control image is taken from an identical assay without primary antibody.

    For other IHC staining systems (automated and non-automated) customers should optimize variable parameters such as antigen retrieval conditions, primary antibody concentration and antibody incubation times.

    *Tissue obtained from the Human Research Tissue Bank, supported by the NIHR Cambridge Biomedical Research Centre

  • Immunocytochemistry/ Immunofluorescence - Goat Anti-Rabbit IgG H&L (Biotin) (ab6720)
    Immunocytochemistry/ Immunofluorescence - Goat Anti-Rabbit IgG H&L (Biotin) (ab6720)Lesage-Padilla, A. et al PLoS One. 2017 Dec 27;12(12):e0189942. doi: 10.1371/journal.pone.0189942. eCollection 2017 Reproduced under the Creative Commons license http://creativecommons.org/licenses/by/4.0/

    ab6720 was used as secondary antobody at 1/1000 dilution for 1 hour at room temperature to detect FOXL2 in mock -transfected and FOXL2-transfected fibroblasts and glandular epithelial cells in ICC/IF. The primary antibody was a Rabbit Anti-FOXL2.

    (From Figure 4B of Lesage-Padilla et al)

  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Goat Anti-Rabbit IgG H&L (Biotin) (ab6720)
    Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Goat Anti-Rabbit IgG H&L (Biotin) (ab6720)This image is courtesy of an anonymous Abreview
    ab28829 (Rabbit polyclonal to STAT6 (phospho Y641)) at 1/100 staining mouse lung tissue sections by IHC-P. The tissue was formaldehyde fixed and blocked with serum. A heat mediated antigen retrieval step was performed prior to incubation with the antibody. Ab6720 (Goat polyclonal to Rabbit IgG H&L (biotin)) was used as the secondary antibody.

    See Abreview

  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Goat Anti-Rabbit IgG H&L (Biotin) (ab6720)
    Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Goat Anti-Rabbit IgG H&L (Biotin) (ab6720)This image is courtesy of an anonymous Abreview.

    ab21156 staining pancreatic amylase in mouse pancreas tissue sections by Immunohistochemistry (IHC-P - paraformaldehyde-fixed, paraffin-embedded sections). Tissue was fixed with paraformaldehyde and blocked with 4% serum for 30 minutes at 25°C; antigen retrieval was by heat mediation in a citrate buffer. Samples were incubated with primary antibody (1/500) for 16 hours at 4°C. A Biotin-conjugated Goat anti-rabbit IgG H&L (ab6720) (1/2000) was used as the secondary antibody.

    See Abreview

  • Immunohistochemistry (Frozen sections) - Goat Anti-Rabbit IgG H&L (Biotin) (ab6720)
    Immunohistochemistry (Frozen sections) - Goat Anti-Rabbit IgG H&L (Biotin) (ab6720)
    Ab6720 was used at dilution 1/100 with the primary antibody ab15000 in IHC-Fr. See the review on ab15000.

Protocols

To our knowledge, customised protocols are not required for this product. Please try the standard protocols listed below and let us know how you get on.

Click here to view the general protocols

References

Publishing research using ab6720? Please let us know so that we can cite the reference in this datasheet.

ab6720 has been referenced in 65 publications.

  • Alexander L  et al. Fractionating Blunted Reward Processing Characteristic of Anhedonia by Over-Activating Primate Subgenual Anterior Cingulate Cortex. Neuron 101:307-320.e6 (2019). PubMed: 30528065
  • Qiao J  et al. Inhibition of HIF-1a restrains fracture healing via regulation of autophagy in a rat model. Exp Ther Med 17:1884-1890 (2019). PubMed: 30783464
  • Lodge EJ  et al. Homeostatic and tumourigenic activity of SOX2+ pituitary stem cells is controlled by the LATS/YAP/TAZ cascade. Elife 8:N/A (2019). PubMed: 30912742
  • Taylor-Whiteley TR  et al. Recapitulating Parkinson's disease pathology in a three-dimensional human neural cell culture model. Dis Model Mech 12:N/A (2019). PubMed: 30926586
  • Verbeeck L  et al. Uncoupling of in-vitro identity of embryonic limb derived skeletal progenitors and their in-vivo bone forming potential. Sci Rep 9:5782 (2019). PubMed: 30962493
View all Publications for this product

Customer reviews and Q&As

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1-10 of 13 Abreviews or Q&A

Question

Dear xxxx,

We would like to order the following antibodies through our Indian distributor:

ab51210 and ab70587.

Request you to provide with the code to take part in the Abtrial testing scheme.

Thanking you,

Regards

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Answer

Thank you for your reply.

I would be pleased to provide you with the Abtrial testing discount codes. In order to do so, could you please confirm which distributor you will be purchasing the antibodies from?

I look forward to receiving your reply.

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Question

Inquiry: Dear Sir/ Madam, I am trying to develop an ELISA protocol with your antibodies. I was looking for scientific support where I could check the application for the three antibodies in the protocol. To further clarify my needs I would like your comment as to whether the three antibodies are compatible.
capture antibody: ab54219
Description Mouse monoclonal [7F12H4,6G5F6] to BTK Host species Mouse
Tested applications Flow Cyt, WB, ELISA, IHC-P, ICC/IF
Cross reactivity Reacts with Human Immunogen Ni-NTA purified recombinant BTK fragment (Human) expressed in E.coli strain BL21 (DE3)

Primary antibody: ab51210 Description Rabbit polyclonal to BTK (phospho Y223) Host species Rabbit
Specificity ab51210 detects endogenous levels of BTK only when phosphorylated at tyrosine 222.
Tested applications ELISA, WB Cross reactivity Reacts with Human Predicted to work with Mouse
Immunogen Synthesized phosphopeptide derived from human BTK around the phosphorylation site of tyrosine 223 (A-L-YP-D-Y).

Detection antibody: ab6720 Description Goat polyclonal Secondary Antibody to Rabbit IgG - H&L (Biotin) (Keeping in mind Alphascreen assay) Host species Goat Target species Rabbit Tested applications WB, ELISA, Immunomicroscopy, Dot Blot, ICC/IF, IHC-Fr, IHC-P Immunogen Rabbit IgG whole molecule
Also could you please let me know whether the first and second antibodies have a common epitope or not. Thanking you, Best regards

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Answer

Thank you for contacting us and your interest in our products.

The antibodies you have chosen for your ELISA should work well together. The immunogen used to raise the mouse monoclonal ab54219 does not cover the Y223 region of BTK and they should therefore not be detecting the same epitope.

However, as we have not tested these antibodies together in a sandwich ELISA we would not be able to guarantee their compatibility. For this reason, you would be eligible to take part in our Abtrial testing scheme.

The way this scheme works is that you would purchase ab54219 and ab51210 as normal and test it in your ELISA. As long as you then share the results with us through submitting an Abreview (no matter whether the results are negative or not) you would then receive the value of ab54219 or ab51210 of a subsequent Abcam order.

More information on this offer can be found from the following link:

https://www.abcam.com/abtrail

The only constraint in this offer is that the antibody to be tested must be purchased, the Abreview submitted, and the free credit claimed within a 4 month period.

If you would like to participate in this offer please do let me know as a discount code needs to be issued prior to the purchasing of either ab54219 or ab51210.

You mentioned that you were setting up an Alphascreen. I am sorry but I did not understand how this would work. Are you just going to use one of the primary antibodies for this? If you could explain this a little more I should be able to help further.

I hope this information has been of help. If I can be of any further assistance, please do not hesitate to let me know.

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Question

Hi Abcam,

Do you mind helping with this enquiry below?

We have found another couple of secondary antibodies on your website that appear identical to AB6788 & AB6720 except for a different concentration.

They are: ab97049 Goat anti-rabbit IgG H&L (biotin)

and ab 97021 Goat polyclonal Secondary antibody to mouse IgG- H&L (Biotin)

What we are hoping to do is to enhance a western blot signal by adding a streptavidin step prior to applying a biotinylated secondary antibody. (This has been successfully done by colleagues in the past).

Would it be possible to get some advice as to which ones would be more suitable for our application?

Thanks for your help :D

Kind regards,

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Answer

Thank you for contacting us and you interest in our products.

There are a few differences between the two pairs of antibodies you have identified. A few of these include the purification used (affitiny vs IgG fraction) as well as the ratio of conjugation. The ab6788 and ab6720 is estimated to have a conjugation ratio of 10-20 per IgG molecule whilst ab97049 and ab97021 have a ratio of 1:4.

I am not familiar with the amplification technique you intend to use and cannot therefore anticipate how these differences may alter the results.

If you could share a more detailed description of the procedure I may be able to advise in more detail.

I hope this information has been of help. If you have any further questions, please do not hesitate to contact us again.

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Question

To whom it may concern,
I would like to know if the antibodies (ab6720 and ab74868) that have been kept at 4C for 2 years could be used for IHC (P).
I'm sure that you are not able to guarantee but I am just wondering if it's worth to try or we shouldn't use them.

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Answer

Thank you for contacting Abcam.

As you mentioned in your email, we at Abcam cannot guarantee that these antibodies would still work after this time, but if have enough samples to run a few test batches to check the validity of the antibodies, then that would definitely be worth trying. If, however your samples are scarce and rather precious, then I would advise playing it safe and purchasing new antibodies.

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

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Question

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:

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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.

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Question

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)

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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.

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Question

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?

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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.

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Question

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,

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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: https://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.

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Question

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,

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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.

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Question

Customer kindly requested Biotin conjugated secondary antibodies for ab2378, ab5690, ab103573, ab10558, ab955, ab53444, ab9018

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Answer

Thank you for your interest in Abcam products. ab2378, ab955 and ab9018 are mouse monoclonal antibodies. I would recommend using ab97044 as a secondary antibody; https://www.abcam.com/Rabbit-polyclonal-Secondary-Antibody-to-Mouse-IgG-H-L-Biotin-ab97044.html ab5690, ab103573, ab10558 are rabbit polyclonal antibodies. I would recommend using ab6720; https://www.abcam.com/Rabbit-IgG-secondary-antibody-H-L-ab6720.html ab53444 is a rat monoclonal antibody. I would recommend using ab6733; https://www.abcam.com/Rat-IgG-secondary-antibody-H-L-ab6733.html To let you know we are running a promotion '4 for 3' on primaries; if you could select 1 more primary antibody then you will be entitled to 8 primary antibodies for the price of 6. Use the discount code and follow the instructions given on website. I hope this information will be helpful. Should you have any other question please do not hesitate to contact me.

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1-10 of 13 Abreviews or Q&A

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