Anti-CCR4 antibody (ab1669)

Merci beaucoup pour toutes ces informations détailléesqui nous sont très utiles pour identifier l'origine du problème rencontré avec le ab1669 lot GR34771-3.

Je n'ai rien à modifier à votre protocole qui me semble être optimal. La quantité de protéines (30µg) est ce que nous recommandons, le test de différentes dilutions d'anticorps primaire ainsi que l'utilisation de différents agents bloquants (lait ou BSA) sont également essentiels; parfait!

Nous n'avons malheureusement pas encore trouvé de publication faisant référence à l'anticorps ab113085. Quant au peptide utilisé comme immunogène, je vais demander au laboratoire produisant cet anticorps si la séquence peut être communiquée.

Je vous contacterai de nouveau dès que j'aurai reçu une réponse.

Merci pour votre patience et n'hésitez pas à m'informer des résultats obtenus avec l'anticorps de remplacement ab113085.

Merci de nous avoir contactés.

Nous sommes désolés d'apprendre que le produit ab1669 que vous avez reçu ne fonctionne pas comme attendu.

Nos produits sont garantis pour fonctionner tels qu'ils sont décrits sur la fiche technique, nous pouvons donc effectivement résoudre le problème que vous avez rencontré en vous envoyant un anticorps de remplacement.

Les 2 anticorps ab83250et ab113085 (comme ab1669) sont garantis pour fonctionner en Western Blot et sur échantillons humains.
L'anti-CCR4 ab83250 n'étant pas actuellement en stock, j'ai mis en place l'envoi d'une unité de ab113085. Le numéro de commande de remplacement gratuit de ce produit est *******. Vous recevrez prochainement un mail de confirmation comprenant les détails d'expédition.

En échange, nous serions ravis si vous pouviez nous communiquer quelques détails concernant votre protocole en remplissant le questionnaire technique ci-joint. Ces informations sont très importantes pour nous car elles nous permettent d'investiguer la source du problème rencontré avec cet anticorps et de prendre les dispositions nécessaires pour assurer une bonne qualité de nos produits.

N'hésitez pas à nous contacter lors d'une prochaine occasion.

Thank you for your enquiry. Ab1667: Antigen Retrieval: Sections were treated with antigen retrieval solution (Dako # S-1700, pH 6.0) and steamed for 20 minutes followed by cooling for 20 minutes. Variations of the method will work successfully as shown in the following publication that has images as well: http://clincancerres.aacrjournals.org/cgi/content/full/11/7/2459 Ab1669: Antigen Retrieval: Sections were treated with antigen retrieval solution (Dako # S-1700, pH 6.0 or #S-3308, pH 8.0) and steamed for 20 minutes followed by cooling for 20 minutes. I hope that this information helps. Should you have any further questions then please do not hesitate to get back in touch with me.

Thank you for your patience. The originator of ab12953 and ab13396 has gotten back to me with the standard IHC protocol that they follow which I have included below. If you have any additional questions, please let me know. Tissue Preparation: Formalin fixation and embedding in paraffin wax Tissue Sectioning: Make 4-µm sections and place on pre-cleaned and charged microscope slides. Heat in a tissue-drying oven for 45 minutes at 60°C. Deparaffinization: Wash dry slides in 3 changes of xylene – 5 minutes each @ RT Rehydration: Wash slides in 3 changes of 100% alcohol – 3 minutes each @ RT Wash slides in 2 changes of 95% alcohol – 3 minutes each @ RT Wash slides in 1 change of 80% alcohol – 3 minutes @ RT Rinse slides in gentle running distilled water – 5 minutes @ RT Antigen retrieval: Steam slides in 0.01 M sodium citrate buffer, pH 6.0 at 99-100°C - 20 minutes Remove from heat and let stand at room temperature in buffer - 20 minutes Rinse in 1X TBS with Tween (TBST) – 1 minute @ RT Immunostaining: (Do not allow tissues to dry at any time during the staining procedure) Apply a universal protein block – 20 minutes @ RT Drain protein block from slides, apply diluted primary antibody – 45 minutes @ RT Rinse slides in 1X TBST - 1 minute @ RT Apply a biotinylated anti-rabbit IgG (H+L) secondary – 30 minutes @ RT Rinse slides in 1X TBST - 1 minute @ RT Apply alkaline phosphatase streptavidin – 30 minutes @ RT Rinse slides in 1X TBST - 1 minute @ RT Apply alkaline phosphatase chromogen substrate – 30 minutes @ RT Wash slides in distilled water – 1 minute @ RT Dehydrate: (This method should only be used if the chromogen substrate is alcohol insoluble (e.g. Vector Red, DAB) Wash slides in 2 changes of 80% alcohol – 1 minute each @ RT Wash slides in 2 changes of 95% alcohol – 1 minute each @ RT Wash slides in 3 changes of 100% alcohol – 1 minute each @ RT Wash slides in 3 changes of xylene – 1 minute each @ RT Apply coverslip

Thank you for your phone call. For ab13268, ab1662, and ab1669, the originator has informed me that the antigen retrieval method for IHC-formalin fixed, paraffin embedded sections involved treating the sections with antigen retrieval solution (Dako # S-1700, pH 6.0 or #S-3308, pH 8.0) and steamed for 20 minutes followed by cooling for 20 minutes. I will contact you again when I receive information regarding ab12953 and ab13396. Have a great weekend!

Thank you for submitting the completed questionnaire to us and for your patience. We are very sorry to hear that you are having problem with this antibody. We have searched our database and found that this is a popular selling product and your feedback is the first we have received about it not working. Therefore, at this stage, we would suggest that there is either a problem with the vial you received, or modifications to your protocol are needed to obtain a positive result. As we understand from your e-mails, you performed IHC on skin biopsies taken from patients with atopic dermatitis. We would strongly suggest using positive control along with the samples. Human spleen sections or human peripheral blood cells would be good for this purpose. This is especially important since there are some publications which suggest that expression of CCR4 in skin-derived lymphocytes is higher that its expression in other type of tissues. Lymphocytes from inflamed skin cells have especially high levels of CCR4 which may be one of the reasons why you are having such a high background signal. Please take a look at this published paper; you may find some useful information: http://ajp.amjpathol.org/cgi/content/full/160/1/347 You may also need to alter certain steps in your protocol. Try to increase the concentration of the blocking serum i.e. 3-5% BSA to see if the background signal is getting lower. Block the endogenous peroxidase activity with 1.6% (not 1%) H2O2, it may help. We have looked at the images you have forwarded to us, it is clear that the antibody works. Comparing the intensity of the signal in the negative control and in the samples, the staining is definitely stronger in the biopsy samples. Although, it is true that the background signal in some areas of the sections is a bit high. We hope this will be useful for you. Should you still have any problem, then please do not hesitate to contact us again.