Anti-CCR7 抗体 (ab1657)

Thank you for your patience. I have been in touch with the source of this antibody once again and I have some additional information. Unfortunately at this time I cannot source an alternative lot. However, the originator mentioned the following valuable information. They mentioned that blocking with 2% rabbit serum should be done prior to incubation with the primary antibody. There is no need for dilution of the antibody in 7.5% rabbit serum. Normal rabbit sera contains low levels of auto antibodies to various antigens in their blood. Normal rabbit serum at such high concentration (7.5%) contains a low level of cross-reacting antibodies that may inhibit binding of goat anti-mCCR7. IgG levels in rabbit serum could be 20-30mg/mL. At 7.5% NRS, the IgG level would be 1.5mg/mL in solution combined with approximately 4 ug/mL of goat anti-mCCR7 antibody. I hope this information helps, if this does not improve the results that you are obtaining I would be happy to provide you with a credit note against this purchase.

Thank you for taking the time to complete our questionaire and contact us. I am sorry to hear you have had difficulty obtaining satisfactory results from this antibody (ab16527). I have checked the procedure used to test this antibody in the laboratory (I have copied this below). Your method follows this very closely, although there are a couple of suggestions I can make to optimise your results. 1. To ensure the sample is not overblocked, I can suggest using just 2% serum for blocking. 2. Can you confirm the constituents of the commercial antigen retreival solution you have used? This antibody has given good results with EDTA buffer heat mediated antigen retrieval for 20 minutes (followed by cooling for 20 minutes). I suggest trying this if you have not done so already. 3. You can increase the signal by increasing the antibody concentration. I suggest trying 1:100. TESTING PROCEDURE: 1.Block the endogenous peroxide by incubating the sections with 3% H2O2 for 15 minutes at room temperature (RT). 2.Wash the sections with PBS and incubate with 2% normal rabbit serum at room temperature to block the non-specific binding sites. 3. Wash the section with PBS and incubate with primary antibody at recommended dilution for 60 minutes at RT. 4. Wash the slides and incubate at RT with biotinylated rabbit anti-goat IgG H+L PUBLISHED PROCEDURE: The following link cites this protocol in the following publication: http://clincancerres.aacrjournals.org/cgi/content/full/11/5/1743 Immunohistochemical Staining. The avidin-biotin complex method was used to detect anti-CCR7, dilution 1:250 Formalin fixed and paraffin-embedded tissues were deparaffinized and subsequently microwaved in EDTA buffer. After preincubation with hydrogen peroxide, avidin/biotin blocking kit, and rabbit serum, the primary antibody was applied for 1 hour at room temperature. After incubation with the secondary antibody (rabbit anti-goat biotinylated), the avidin-biotin complex was added and the enzyme activity visualized with diaminobenzidine. Counterstaining was done with hematoxylin. I hope this information is helpful. Should the suggestions not improve the results, please do not hesitate to contact me again.

Thank you for your enquiry. I am sorry to hear that you have been obtaining non-specific staining using this antibody. I have read your technical questionnaire and I have a few comments. Please can you detail the duration of antigen retrieval that you have been performing. We make the recommendation that antigen retrieval is performed for 20 minute as detailed on the antibody datasheet. My concern is that the tissue preparation is being compromised influencing the presentation of the epitope to the antibody thereby causing non-specific staining. I would also appreciate comments with regards whether you have tried no antibody control experiments to control for non-specific staining of this antibody. Secondary antibodies are often discounted as being causal of non-specific staining. I look forward to hearing from you.

Thank you for your enquiry. Further to our telephone conversation yesterday afternoon I have been in touch with the source of this antibody. They informed me that by western blotting an actual molecular weight of 43KDa was detected in the following publication. I will e-mail this reference in a follow up e-mail. Walker SR et al. Murine neuroblastoma attenuates dendritic cell cysteine cysteine receptor 7 (CCR7) expression. J Pediatr Surg 40:983-7 (2005). PubMed: 15991182 It may be useful to examine the western blotting approach that was employed in this publication. However, following close examination of this reference it is clear that they do not show a molecular weight standard and the band is cropped in the image, therefore making it difficult to know the ACTUAL migration of the band. Should you continue to have difficulty with this antibody I would encourage you to submit a technical questionnaire by clicking on the link below. This will better enable our technical team to determine the steps that you have taken to optimise this antibody. https://www.abcam.com/index.html?section=western&pageconfig=technical&intAbID=1657&mode=questionaire I hope this information helps, please do not hesitate to contact us if you need any more advice or information.

Thank you for your e-mail. We hope these answers are more helpful. 5. How did you block the unspecific binding sites? BSA 3% 30 minutes Instead, use 2% normal rabbit serum at room temperature to block the non-specific binding sites. 7. Secondary antibody ? What secondary antibody are you using? Specification (in which species was it raised against)? At what dilution(s) have you tested this antibody? Incubation, wash steps? Do you know whether the problems you are experiencing come from the secondary? What detection method are you using? Antigoat secondary antibody 1:400. Try to apply an ABC signal enhancing step. Wash the slides and incubate at RT with biotinylated rabbit anti-goat IgG H+L at 1:200 dilution for 30 minutes. Wash the slides and incubate with HRP-Streptavidin 1:400 dilution for 30 minutes. Wash the slides and incubate with DAB for 5-7 minutes at RT. Wash slides with PBS and counter stain with hematoxylin.

We are very sorry you are having problems with your protocol. Here is the up to date detailed protocol: .