Anti-F4/80 antibody [BM8] (ab16911)

Estimada ,
Agradecemos mucho tus sugerencias. Sin embargo hemos intentado muchas
de ellas también sin éxito. Te las enumero a continuación:
- Hemos bloqueado la biotina endógena (no en el caso del hígado pero
sí en los tumores).
- Hemos usado tanto BSA como suero de la especie en la que se generó
el secundario (hemos usado un donkey anti rat como 2rio, por lo que
empleamos donkey serum)
- No usamos permeabilizadores porque el antigen retrieval en principio
suele ser suficiente para que el anticuerpo penetre en las secciones,
sobre todo tratándose de una proteína de membrana.
Además, hemos realizado varias pruebas con diferentes protocolos para
la recuperación antigénica (que es quizá el paso más determinante),
Te agradecería mucho si pudieras mandarnos directamente el anticuerpo de
reemplazo.
Muchas gracias de nuevo por tu respuesta.
Un saludo,

ANSWER:

Gracias por tu respuesta.

No hay ningún problema, acabo de realizar el nuevo pedido, completamente gratuito de ab16911, con número de pedido XXXXX. No olvidéis mencionar este código si tenéis que contactar con nuestro Servicio al Cliente. En principio el anticuerpo os llegará mañana.

Recibiréis una confirmación automática del mismo informando sobre la fecha estimada de entrega y detalles del pedido.

Espero que este nuevo anticuerpo os funcione perfectamente. En cualquier caso, si volvéis a usar tejido de hígado yo os recomendaría encarecidamente bloquear la biotina endógena, presente en elevadas cantidades en este tejido, y que en caso de no bloquearla, es muy posible que os de una tinción con mucho fondo.

Lamento mucho las molestias ocasionadas, y quedo a vuestra disposición para cualquier otra duda o información que pudierais necesitar.

What does the note "The protein is sensitive to beta-mercaptoethanol" on the datasheet for this antibody mean? Should I run my samples reduced or non-reduced?

ANSWER:

Thank you for contacting us.

Only non-reduced reduced samples should be used to detect F4/80.

If you have further questions please do not hesitate to contact us.

I have been using the rat monoclonal BM8 to F4/80 ab16911 and was wondering if you could tell me whether the antibody binds a surface or intracellular epitope of the immunogen?

Also, is the peptide available to use as a preabsorbed control?

This would be extremely helpful to know...

Thanks

ANSWER:

I regret to inform you we have no information about the epitope recognized by the antibody. So we do not know if the antibody recognizes a surface or intracellular epitope of the immunogen. The peptide is not available unfortunately.

I'm sorry I couldn't help you more on this occasion.

Is there a protocol for the proteinase antigen retrieval method that is recommended? Can microwave antigen retrieval be done instead?

ANSWER:

Thank you for your enquiry. Our source for this antibody advises using the proteinase K antigen retrieval method. Enzymatic digestion with Proteinase K in 0.05 M Tris-HCl, 15 mM sodium azide, pH 7.5. (3-12 minutes at room temperature).

A full protocol for this method can be found at the website IHC world: http://www.ihcworld.com/_protocols/epitope_retrieval/proteinase-k.htm

Also, the following reference describes the use of [BM8] in IHC on paraffin embedded sections:

Stevceva L et al. The inflammatory infiltrate in the acute stage of the dextran sulphate sodium induced colitis: B cell response differs depending on the percentage of DSS used to induce it. BMC Clin Pathol 1:3 (2001). PubMed: 11580872

Please contact us again if you have any additional questions.

BATCH NUMBER 156930 ORDER NUMBER PO#10413

DESCRIPTION OF THE PROBLEM No staining

SAMPLE mouse lung tissue

PRIMARY ANTIBODY 1:50-1:200 several dilutions in PBS with 1%BSA (overnight at 4?C) Rinse for five minutes 3 times with PBS

DETECTION METHOD ABC-Fluorescent

POSITIVE AND NEGATIVE CONTROLS USED CD45 antibody from [a competitor] worked great under the same conditions

ANTIBODY STORAGE CONDITIONS Stored at -20?C

FIXATION OF SAMPLE Pormalin-fixed paraffin-embedded

ANTIGEN RETRIEVAL Heat mediated: microwave method (15 minutes)

BLOCKING CONDITIONS hydrogen peroxidase for half hour and normal goat serum for blocking

SECONDARY ANTIBODY biotinylated Goat anti-Rat IgG Rinse for five minutes 3 times with PBS

HOW MANY TIMES HAVE YOU TRIED THE APPLICATION? 3 HAVE YOU RUN A "NO PRIMARY" CONTROL? Yes DO YOU OBTAIN THE SAME RESULTS EVERY TIME? Yes

ANSWER:

I'm sorry to hear one of your customers is having problems with ab16911.

There may be several reasons for the problem:

-the antibody was stored at -20C and should be stored at 4C. It may therefore have been damaged because of this storage.

-The antibody required proteinase treatment of the sections, as detailed on the datasheet. Heat mediated antigen retrieval may not work. Please follow our recommendation.

-The antibody is applied too dilute. Please try 1:10 incubation and more dilute too.

-the secondary antibodies does not work well or is not applied long enough. We typically recommend a minimum of 1 hour incubation. Please ask the customer to check that the secondary and ABC/ fluorescent detection kit work well.

Please let me know if I can be of further help,