Anti-CD68 antibody [KP1] (ab955)

Hola ,
¿Qué tal? estoy interesada en inmunofluorescencia
de doble marcaje en tejidos humanos parafinados.
En concreto me interesarían los anticuerpos primarios, a ser posible
monoclonales, de distintas especies para poder combinarlos, contra las
siguientes proteínas humanas:
* OCT4: ab18976, ab109183, ab129030
* tenascin C: ab6346, ab3970, ab108930, ab86182
* CD68: ab955, ab845, ab125047
* S100A6: ab11181, ab117714, ab75676
* ganglioside GD2: ab68456
* Sox10: ab107532, ab122977
* MMP9: ab76003, ab119906
* CD163: ab111250, ab100909
* PNMT: ab119784
He indicado los productos que me parecían interesantes de la web, pero
acepto sugerencias.
Y tambíen estaría interesada en los anticuerpos secundarios marcados
con distintos fluorocromos para anticuerpos de distintas especies.
Necesitaría la ficha técnica y el precio de cada uno de ellos.
Muchas gracias.
Saludos,

ANSWER:

Muchas gracias por tu respuesta.

Es importante que todos los anticuerpos que escojas para llevar a cabo IHC-P en tejido humano estén validados (y por tanto garantizados por Abcam) para funcionar en esta especie y aplicación. Esta información aparece en la hoja técnica de los productos.

También es importante echar un vistazo a las hojas de datos de cada uno de los productos para tener en cuenta recomendaciones especificas, tales como diluciones, métodos de recuperación antigénica, y demás que pueden aparecer en ellas. A las fichas técnicas se puede acceder desde la página web, escribiendo el código del anticuerpo (ab955, por ejemplo) en la casilla azul en la parte de arriba de nuestra página web (http://www.abcam.com/).

Desconozco las combinaciones que queréis llevar a cabo mediante inmunofluorescencia doble con los anticuerpos mencionados. En general, a la hora de hacer inmunoensayos múltiples hay que tener mucha precaución para evitar reactividad cruzada. Es importante usar anticuerpos procedentes de distintas especies si se puede, o al menos con diferentes isotipos. Una buena forma de evitar reactividad cruzada es usando secundarios de la misma especie. Además esto permite usar suero de dicha especie para bloquear.

Te copio los links a los protocolos que tenemos en nuestra web para llevar a cabo doble inmunofluorescencia por s pudieran resultar de ayuda:

http://www.abcam.com/index.html?pageconfig=resource&rid=11459

http://www.abcam.com/index.html?pageconfig=resource&rid=11458

De todas maneras si quieres que comentemos distintas posibilidades y escenarios posibles, no dudes en contactarme otra vez.

Respecto a los secundarios, tenemos muchos anticuerpos que podrían serviros. Te aconsejo que en función de los primarios y combinaciones que vayáis a llevar a cabo, optéis por uno u otro. Para llevar a cabo la búsqueda de los secundarios desde la pagina web (http://www.abcam.com/) pincha en la pestaña “Secondary Antibodies”. Se abre una nueva ventana de “Advance Search”, en la cual se pueden elegir las categorías requeridas. La única categoría obligatoria es el isotipo del anticuerpo, pues tiene que coincidir con el del primario. El resto de categorías son opcionales, pero son muy útiles para afinar la búsqueda y elegir el anticuerpo más apropiado según la especie del primario, la conjugación deseada, la clonalidad, la aplicación en la que se va a testar, etc…

Los precios y la disponibilidad pueden consultarse igualmente en la parte superior de la datasheet de los productos. Si necesitas un presupuesto no dudes en hacérmelo saber para que te lo envíe. Además tenemos algún descuento para pedidos superiores a 5 productos, por lo que si vais a realizar un pedido de estas características te animaría a que nos contactaras primero.

Espero que esta información sea de ayuda. Como te digo, para cualquier aclaración o más sugerencias, estaré encantada de echar una mano.

Hi Abcam,
I have received the following enquiry from a customer, please see below;
AB955 – Anti-CD68
AB81720 – Anti-CD73
AB95139 – Anti-VCAM1
AB6630 – Anti-CD62E (E-selectin)
I have also read the fact sheets for all, but to clarify, they are all suitable for IHC using frozen sections fixed with PFA?
Could you please confirm the above?
Thanks for your help!
Kind regards,

ANSWER:

Thank you for contacting us.

Effectively all of the products mentioned are suitable, and therefore are guaranteed for IHC in frozen sections in the species stated on their datasheets.

However, instead of using PFA as a fixative, I would better suggest using acetone, methanol or ethanol at -20°C for 30 min, so the additional antigen retrieval step can be skipped. If, by the tissue’s nature PFA is required as a fixative, antigen retrieval is recommended to allow the antibody reach the epitope.

All of our products are covered by our Abpromise® guarantee, which ensures that you can trust them, 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. Please do not hesitate to contact us if you need any more advice or information.

1) Abcam product code: ab8330, ab7388, and ab955
2) Abcam order reference number: XXXXXX
3) Problem: lack of signal compared to no-primary control
4) Sample prep: mouse brain, heart, lung, IHC-P for positive control; negative control is same tissue with no primary antibody
5) Fixation step: yes, 10% formaldehyde fix, 10-20 minutes room temperature
6) Antigen retrieval method: sodium citrate buffer (1.92g citric acid, 1N NaOH, 1000mL water, 0.5mL Tween-20 pH 6.0) at 95*C for 30 minutes, allow to cool for 45 minutes
7) Permeabilization: There is .05% Tween-20 in the antigen retrieval step and 0.1% Triton X-100 in the block step
8) Block: 1% BSA + 0.1% Triton X-100 in HBSS, 30 minutes, room temp
9) Endogenous peroxidases and biotins blocked? No
10) Primary antibodies: ab8330 at 1/1000, ab7388 at 1/250, and ab955 at 1/200, all in the block buffer listed above, for 1 hour room temp.
11) Secondary antibodies: Molecular Probes goat anti-mouse IgG alexa fluor 568, Molecular Probes A11077 goat anti-rat IgG alexa fluor 568, 1/1000 in block buffer listed above, for 1 hour
12) Washes: yes, there are 3x 5-minute washes after the primary and secondary antibody steps
13) Detection method: fluorescence using a Nikon confocal microscope
14) I have tried this particular staining once using established protocols in the lab. Since Ab7817 worked fine, I am confident with the protocol.

Thanks!

ANSWER:

Thank you for taking time to complete our questionnaire. I am sorry to hear that this antibody is not providing satisfactory results. The details provided will enable us to investigate this case and will provide us with vital information for monitoring product quality.

Having reviewed this case, I would like to offer some suggestions to help optimize the results from ab955 since this antibody has been tested in mouse paraffin-embedded tissue sections.


For the fixation, we generally recommend between 18 - 24 hrs. It may be just that your sections are underfixed.


For antigen retrieval, was this performed in the microwave? We generally recommend 20 mins, but it's best to optimize between 15 - 30 mins. When the 20 mins has elapsed, you can run cold tap water into the vessel for 10 mins.


If you want to permeablize the sections, you should do a 0.1% Triton-X 100 in PBS treatment as a separate step and omit the Triton X-100 from the blocking buffer.


For blocking, have you tried5-10% normal goat serum instead of BSA? You should also put this in PBS.


The most important thing to try wouldbe to test a1/100 dilution of ab955 overnight at 4C instead of 1/200 for 1 hr RT. You should do this incubation in 1% BSA in PBS.


For the s100 antibody ab8330, would you be able to run a positive control with rat tissues since this antibody isn't tested in mouse?


For the CD31 antibody, ab7388, we know that this does not work well with PFA fixation (see Abreview linkedbelow). We have heard that it works wellwhen used on frozen sections of mouse skin that werefixed in methanol:acetone for 10 mins. Would you be able to try some frozen methanol or acetone fixed sections as a positive control since IHC-P does not work.


http://www.abcam.com/index.html?datasheet=7388&tab=abreviews&intabreviewid=23226

Should the suggestions not improve the results, please do let me know.

In the event that a ab955 is not functioning in the species and applications cited on the product datasheet (and the problem has been reported within 6 months of purchase), we would be pleased to provide a free of charge replacement, credit note, or refund for this tested antibody.

I hope this information is helpful, and I thank you for your cooperation.

I was wondering if you know if ab955 (anti-CD68 amtibody [KP1]) can be used for Western blotting? Thanks for your help.

ANSWER:

Thank you very much for your interest in anti-CD68 antibody clone [KP1] (ab955).

To our knowledge, ab955 has not been tested in Western blot, but I do not see a reason why it should not work. Therefore, I can offer a discount off a future purchase if you buy ab955 now, test it in Western blot and submit feedback to us in the form of an Abreview. It doesn’t matter whether the Abreview is positive or negative, we would just really like to receive your feedback. The discount would be to the value of 1 free primary antibody.

If you are interested in this offer, please follow these steps:

1. Reply to this e-mail to let me know that you would like to proceed and test ab955 in Western blot. I will then send a discount code. This code must be issued before purchasing ab955 so please wait for my reply before ordering.

2. Purchase ab955 either by phone, fax, or online (www.abcam.com).

3. Test it in Western blot.

4. Let us know the results, positive or negative, using our Abreview system (this will take about 10 minutes and images are great if you have them!). To find out more about our Abreview system, please visit: http://www.abcam.com/abreviews.

5. After the review is submitted to us, the discount code becomes active. Simply place your new order by phone, fax, or on the web and mention the discount code. The discount can be redeemed for any primary antibody ordered and the discount code is valid for 4 months after issue.

We are always pleased to obtain feedback about our products and any information is greatly appreciated! Even if ab955 turns out to be unsuitable for Western blot, you will still receive the discount on your next purchase after your Abreview has been submitted.

Please let me know if you have any questions about this offer and I would be happy to help you further.

The Terms and Conditions of this offer can be found at: www.abcam.com/collaborationdiscount

I am interested in purchasing a secondary antibody for use in immunohistochemistry that can work with the following primary antibodies from your company (ab24590, ab7817, ab955, and ab89064). I have searched the catalog and found ab6728 and ab6788 as possible candidates for this purpose. Could you kindly suggest on which one would work better with those primary antibodies for immunohistochemistry (IHC-P)?

Thank you,

ANSWER:

Thank you for contacting Abcam.

I feel that both products are suitable for IHC-P. Both have many specific references and in-depth scientific support data right on the datasheet. They differ mainly in the species in which they were raised and in the tags used for detection. Ab6728 is an HRP conjugate whereas Ab6788 is Biotin labeled. I believe that you should choose the secondary which has an associated detection system that you are most familiar with in case you should run into any staining issues.

I hope this information is helpful. Please let me know if you have any questions.