Mouse and Rabbit Specific HRP/DAB detection IHC kit (ab64264)

Inquiry: i am using ab292 for staining bone marrow section formalin fixed parrafin embedded . i read the protocol used for the human adrenal gland found attached to the antibody sheet .i have some questions please: 1-if i am going to use the ab64264 as detection kit,should i have to do the blocking step with serum for 30 min as written in adrenal gland protocol and in general abcam protocol or the protein block in the detection kit for 5 min can replace this step? 2-can i perform the antibody dilution using the ready to use antibody diluent or i should prepare the diluent as in abcam general protocol? thanks alot for support

ANSWER:

Thank you for contacting us.

The protocol mentioned described on the datasheet refers to an Abreview from a previous user of this antibody. Different protocols may also work.

If you are using ab64264 kit, there is no need to use an extra blocking agent, as the kit already contains a protein block buffer to avoid unspecific bindings.

Regarding the antibody dilution, you can either use the ready to use antibody diluent or the general recommended diluent buffer (1% BSA in PBST).

You can use our online protocols as a general guidance:

http://www.abcam.com/index.html?pageconfig=resource&rid=11384

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

Dear Sir or Madame,

Could you please inform me, whether is possible to order a DAB substrate alone in the Mouse and Rabbit specific HRP/DAB detection IHC kit (ab64264)?

Thank you very much

ANSWER:

Thank you for your enquiry which has been forwarded to the Scientific support team.

I am sorry, it is regrettably not possible toprovide this particular component separately.

If you have any further questions, please do not hesitate to contact me.

Dear All,



Please let us know about the following that many slides should have been available for the concentrated antibodies after dilution and how many slides can be prepeared with the prediluted slides. Also let us know the about the deteation kit and DAB ab64264 and



ab3223

ab63340

ab74179

ab8105

ab27595 (pre-diluted)

ab27596 (pre-diluted)

ab15467 (pre-diluted)


ab64264 -sufficient for how many slides?

Awaiting your early response.

Sincere regards,

ANSWER:

Thank you for your inquiry.

Since the working dilutions of antibodies are not a fixed value but have to optimised by the endusers themselves according to experimental condition, I cannot give you absolute numbers. Even when the pre-diluted antibodies are used neat there might be reasons to use 50ul per slide or maybe even 100ul per slide.

These conditions include: size of the sample, tissue type, lab experience, diluent buffer, species, antigen retrieval, incubation time, etc.

For the products you mentioned I can give a estimate for the pre-diluted antibodies of ca 140- 280slides, for the other antibodies 500 - 100 slides and for the detection kit 150 - 300 slides.

I hope this information is helpful.

Dear Sir or Madame,

I have two questions regarding your protocol for your Polyvalent HRP/DAB detection kit.

1.Does the antigen retrieval (TE buffer) has to be performed before adding Hydrogen Peroxide block?

2. In your IHC-P protocol you suggest blocking of endogenous peroxidase after primary antibody incubation, but in ab64264 protocol this is advised as step 5 after Hydrogen peroxide block. Which is better?



Thank you very much in advance!

ANSWER:

Thank you for your inquiry.

The protocol for ab64264 reflects the sequence we follow in house and this kit is tested towork useddoing the steps in this order.

Personally I have done the endogenous peroxidase blocking step before antigen retrieval, after antigen retrieval and after the primary antibody and my sense is that it doesn’t matter whThank you for your inquiry.

The protocol for ab64264 reflects the sequence we follow in house and this kit is tested to work used doing the steps in this order.

Personally I have done the endogenous peroxidase blocking step before antigen retrieval, after antigen retrieval and after the primary antibody and my sense is that it doesn’t matter when as long as it is before the HRP step. I do not have any experimental data to support that statement but that is my personal opinion.

Please do not hesitate to contact me with additional questions or concerns.
en as long as it is before the HRP step. I do not have any experimental data to support that statement but that is my personal opinion.

Please do not hesitate to contact me with additional questions or concerns.

 We have a customer who is interested in the above kit, but he is using fresh frozen samples and not fixed paraffin. He has suggested if he just removes the first step on the protocol, it should work for him. What is your opinion, will he run into any problems? He is also wondering how many reactions will he get out 1 kit (15ML)? I look forward to your response. Kind Regards    

ANSWER:

Thank you for contacting us.

Theoratically, this kit can be used for IHC-Fr also. Please do not follow first step in protocol. For other steps please follow the protocol as required.

The number of reactions depends on the use of this kit. There is 15 ml solution of each component except DAB chromogen (0.5ml, 50X) which will be suitable for many reactions based on the surface area of tissue sections. Please be advised that 1ml volume informaiton on the datasheet is incoprrect at the moment, we will be updating it shortly.

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