Anti-beta 2 Microglobulin antibody (ab15976)

Dear Madam or Sir,

I am searching for an antibody to stain paraffin-embedded xenografts by IHC.

I found in your catalog the following antibodies:

ab15976

ab58890

ab54810

ab80071

ab70328

Could you maybe tell me which of these antibody would work best for my purposes?

Sincerly,

ANSWER:

  Thank you for contacting us. For detecting anti-beta 2 Microglobulin with IHC-P we have five products in our catalogue, all tested in Human. http://www.abcam.com/index.html?pageconfig=searchresults&search=beta 2 Microglobulin&pt=1&sk=app&sv=74&sn=IHC-P&l=2&fViewMore=1 Ab15976 contains a positive Abreview from a customer who tested it in ICC/IF obtaining satisfactory results. The difference between the antibodies lies in the clonality and the species they were raised in. So, depending on the species you need to test, you would prefer an antibody rose in mouse or in rabbit in order to minimize rejection between species. For targeting HLA Class 1 ABC I’m afraid the only option available is ab70328. In any case, all of our products are covered by our Abpromise® guarantee, which ensures that you can trust our products, and they should work in the tested species and applications stated on the datasheet, or we will offer a replacement, credit, or refund, if reported within 6 months of purchase. To read about our Abpromise policy: http://www.abcam.com/index.html?pageconfig=abpromise I hope this information is helpful to you. If you have any questions or if you need further help or a proforma quote then please do not hesitate to contact me.

BATCH NUMBER 107044 ORDER NUMBER 78951

ANTIBODY STORAGE CONDITIONS I am just wondering what the concentration of this batch of AB15976 is? In the datasheet, the recommended dilution for ELISA (as coating antibody) is 10?g/ml, but I can't find the concentration of the product anywhere on the datasheet?

ANSWER:

The protein concentration for this batch number is is 11.0 mg/ml.

BATCH NUMBER 107044 ORDER NUMBER -- NOT SPECIFIED --

WHAT STEPS HAVE YOU ALTERED? The customer asks what would be your recommendation to minimize these xreativities.

ADDITIONAL NOTES CUSTOMER COMPLAINT FORM REKLAMATIONSFORMULAR

PRODUCT DETAILS / PRODUKT-MERKMALE:

Product code / Katalog-Nummer: ab15976

Product description / Produkt-Bezeichnung: Anti-beta-2-Microglobulin Batch number / Lot-Nummer: 107044 Date received / Eingangs-Datum: 28.04.05

Date first used / Anbruchs-Datum: 11.05.05

Storage conditions used / Lagerungsbedingungen: +4?C

Customer complaint (enter details in attached trouble shooting formular) / Reklamationsgrund / Problemschilderung (bitte machen Sie in dem anh?ngenden Fehlersuche-Fragebogen genaue Angaben):

unspecific binding to mouse tissue

for my doctoral thesis I have to detect mesenchymial stem cells 8hMSC) on a anatomical structure after implantation into mouse. This is why the ab has to detect only the human antigen w/o crossreacting with mouse. The immunofluorescence labelling has been performed on the one hand with OT with hMSC and on the other hand on this anatomical structure after explantation from mouse (with connective tissue that has been grown) . The anti b2 Microglobulin ab showed unspecific binding to mouse tissue.

Unspezifische Bindung an das Mausgewebe. In meiner Doktorarbeit mu? ich die humane mesenchimale Stammzellen (hMSC) auf den Leitschienen nach der Implantation in die Maus nachweisen. Deswegen mu? der Antik?rper an die humane Zellen binden, darf aber nicht mit dem Mauisgewebe nicht reagieren. Die Immunfluoreszenzf?rbung wurde einmal an den mit hMSC besiedelten OT durchgef?hrt und einmal an den Leitschienen nach der Explantation aus der Maus (eingewachsenes Bindegewebe). Anti-beta-2-Microglobulin zeigt eine unspezifische Bindung an das Mausgewebe.

Did you repeat the experiment: / Wurde das Experiment von Ihnen wiederholt? NEIN/No

Did you us this product before / Haben Sie das Produkt fr?her verwendet: NEIN/No TROUBLESHOOTING SHEET FRAGEBOGEN ZUR FEHLERSUCHE

TISSUE/CELL PREPARATION USED / ANGABEN ZUR GEWEBS- / ZELL-PR?PARATION:

Species / Spezies: Maus/Mouse Strain / Stamm: atyme Nacktm?use/Nude Mice

Sample type: Frozen section / paraffin embedded / cytospin / cell smear / Art der Probe:

Gefrier-Schnitt/Cryo section

Tissue used / verwendetes Gewebe:

Leitschiene nach der Explantation aus der Maus (eingewachsenes Bindegewebe) I can?t translate Leitschiene, it?s obviously a particular anatomical structure from mouse with connective tissue

Fixation details (please provide full details) / Gewebe-Fixierung (machen Sie m?glichst detaillierte Angaben): 100% Methanol 8min bei/at -20?C Tissue pre-treatment (please provide full details) / Gewebe-Vorbehandlung (machen Sie m?glichst detaillierte Angaben):

Keine Vorbehandlung no pretreatment

STAINING PROTOCOL / ANF?RBUNGS-PROTOKOLL:

Primary antibody details / Angaben zum prim?ren Antik?rper:

Anti-beta-2-Microglobulin

Dilution / Verd?nnung: 1:200

Volume / Volumen: 1ml

Incubation time / Inkubationszeit:

1St. Raumtemperatur, ?ber Nacht im K?hlschrank bei +4?C 1 h RT, ON at 4?C in the fridge

Secondary antibody details / Angaben zum sekund?ren Antik?rper:

Alexa Fluor 488 goat anti-rabbit IgG higly cross-adsorbed

Dilution / Verd?nnung: 1:200

Volume / Volumen: 1ml

Incubation time / Inkubationszeit: 1St. /1 h

Temperature / Temperatur: Raumtemperatur/RT

Other (please add any other relevant details, including use of blocking sera etc) / Sonstiges (bitte erg?nzen Sie alle relevanten Einzelheiten, z.B. den Gebrauch von Blockierungs-Serum etc.):

1St. Blockierung mit dem Ziegenserum vor dem Prim?rantik?rper Blocking 1 h with goat serum

Details of controls used / Einzelheiten zu den durchgef?hrten Kontrollen:

Please give details of negative and/or positive controls that have been used / Bitte machen Sie Angaben zu den Negativ- und/oder Positiv-Kontrollen, die verwendet wurden:

Negativ-Kontrolle: auslassen von Prim?rantik?rpern negative control w/o primary abs

ANSWER:

I'm sorry to hear you are having a problem with ab15976.

This antibody has only been tested in IHC using formalin fixed, paraffin embedded human tissue. It may very well cross-react with mouse, and it has never been tested on frozen sections or on cytospins, so we do not know if it will work in these applications.

Keeping this in mind, however, I would like to suggest the following modifications to your protocol that I believe may reduce this binding if it is non-specific: 1) please try blocking endogenous peroxidases by using 0.3% H2O2 in PBS for 10 minutes before adding the primary antibody, 2) try diluting the antibody to 1:800 and incubating for 30 minutes at room temp, 3) block with 10% normal goat serum, and 4) try a different fixation such as formalin, PFA, methanol or acetone.

Please let me know if this helps and do not hesitate to contact us for further advice,