Biotinylated Goat anti Mouse IgG (H+L) (Ready to Use) (ab64255)

I ordered the product Biotinylated Goat anti Mouse IgG (H+L) (Ready to Use) (ab64255) some time ago

and I just wanted to ask, if it is intended to use the antibody without diluting it with buffer, because

I’m missing information concerning the concentration of this product.

Sincerely

ANSWER:

Thank you for contacting us.

This is a ready to use product. You do not need to dilute it further.

However if you get high intensity of staining then you may dilute it 1/10, this way you can do more tests and antibody will last longer. Please be advised that this is just an advice based onmy own research experience.

I hope this information is helpful to you. Please do not hesitate to contact us if you need any more advice or information.

I need to develop double IHC analysis on formalin-fixed paraffin-embedded (FFPE) tissue sections of human tumors specimens, and after we have to do laser capture microdissection of positive cells.   I already have two primary antibodies directed against CD44 (produced in mouse) and against ALDH1 (produced in rabbit).   I'm looking for double staining kit (or all of the individual components which analysis needs) with alcohol insoluble chromogens because after IHC and before laser cature microdissection I have to dehydrate the slides.   Thank you for your time and consideration.

ANSWER:

Thank you for contacting us.

We do not currently offer double staining IHC kits. Your staining conditions are challenging as, of the two common HRP chromogens, only DAB is alcohol insoluable. While of the common AP chromagens, only BCIP/NBT is insoluable. This means that to do double labeling you will have to do a sequential stain first using one detection system and then with the other.

I have collected a list, with links, of the reagents which you would need for this type of staining.

1) After de-parrafinizing and re-hydrating the samples, perform an antigen retrieval step treating with Cirate buffer pH6.0,http://www.abcam.com/10x-Citrate-Buffer-pH-6-0-ab64214.html, in the microwave or pressure cooker. After performing the antigen retrieval rinse in cold tap water for 10 minutes.

2) Rinse slides in TBST,http://www.abcam.com/20x-TBS-T-with-Tween-20-ab64250.html.

3) Performing a blocking step using protein block,http://www.abcam.com/Protein-Block-ab64226.html.

4) Incubate sections with your first primary antibody, for clarity I will assume the CD44 antibody, raised in mouse.

5) Wash with TBST.

6) Block endogenous peroxidases using aHydrogen Peroxide Blocking Reagent,http://www.abcam.com/Hydrogen-Peroxide-Blocking-Reagent-ab94666.html.

7) Incubate your tissue with a biotinylated secondary antibody, in this instance anti-Mouse IgG,http://www.abcam.com/Biotinylated-Goat-anti-Mouse-IgG-H-L-Ready-to-Use-ab64255.html.

8) Wash with TBST.

9) Detect your first antibody usingStreptavidin Peroxidase (Ready to Use)http://www.abcam.com/Streptavidin-Peroxidase-Ready-to-Use-ab64269.html. Note that this does not containa serum or BSA solution as diluent. Serum may contain biotin therefore competing Streptavidin binding with biotinylated secondary antibody, therefore reducing binding activity.

10) Apply your chromogen, DABsubtrate,http://www.abcam.com/DAB-Substrate-Kit-ab64238.html.

11) Wash with TBST.

12)Performing a second blocking step using protein block,http://www.abcam.com/Protein-Block-ab64226.html.

13)Wash with TBST.

14)Incubate sections with the your second primary antibody, this time ALDH1, raised in rabbit.

15)Wash with TBST.

16)Incubate your tissue with a biotinylated secondary antibody, in this instance anti-Rabbit IgG,http://www.abcam.com/Biotinylated-Goat-Anti-Rabbit-IgG-H-L-Ready-to-use-ab64256.html

17)Wash with TBST.

18)Detect your first antibody usingStreptavidin Alkaline Phosphatase (Ready to Use)http://www.abcam.com/Streptavidin-Alkaline-Phosphatase-Ready-to-Use-ab64268.html.

19) Apply your second chromogenAlkaline Phosphatase chromogen (BCIP/NBT) - Ready to Use,http://www.abcam.com/Alkaline-Phosphatase-chromogen-BCIP-NBT-Ready-to-Use-ab7468.html.

18) Wash with TBST, counterstain if desired.


I hope this information is helpful to you. Please do not hesitate to contact us if you need any more advice or information.

1) wie rein ist ab64255 (BSA?)? Ich moechte den AK radiomarkieren, biotin auch wichtig. 2) Konzentration/Volumen? "125ml at mg/ml" macht keinen Sinn.

ANSWER:

Vielen Dank für Ihren Anruf.

ab64255 wurde über Affinitätschromatographie aus dem Antiserum aufgereinigt; der exakte Reinheitsgrade wurde leider nicht bestimmt. Der Puffer, in dem der Antikörper vorliegt, enthält BSA. Die Konzentration beträgt 7 µg/ml, und weil es sich um ein ready-to-use-Produkt handelt, bekommen Sie tatsächlich 125 ml !

Für Ihre Versuchsvorhaben könnte sich dieser Antikörper daher vielleicht nicht unbedingt eignen - weil sich 125 ml doch eher schlecht verarbeiten lassen. Gerne möchte ich Ihnen zwei Alternativen nennen, die in weniger Volumen erhältlich sind (ab97021 und ab97033). Beide Antikörper sind biotinyliert, ab97033 ist zusätzlich noch prae-absorbiert, d.h. er reagiert nicht mit den auf dem Datenblatt angegebenen Spezies (wie Huhn, Rind, Ziege etc): Click here (or use the following: http://www.abcam.com/index.html?datasheet=97021). Click here (or use the following: http://www.abcam.com/index.html?datasheet=97033).

Ich hoffe, diese Informationen helfen Ihnen weiter. Bitte zögern Sie nicht, sich wieder bei uns zu melden, falls Sie weitere Fragen haben.