Rabbit specific HRP/DAB (ABC) Detection IHC Kit (ab64261)

This issue may be result of sample preparation. It is oftentimes the case that the slides have not been inadequate deparaffinization which will lead to this type of staining. The link below outlines Abcam recommendations of deparaffinization: https://www.abcam.com/index.html?pageconfig=resource&rid=11487 Another cause can be insufficient fixation. If formalin has not had sufficient time to penetrate and fix the tissue, there will be uneven fixation, and as a result uneven staining.

One drop of DAB chromogen is about 30 ul, and it is to be added to 1.5 ml of the substrate (50 drops). So the ratio is 1:50.

The mixture cannot be stored very long once you have made it. Ideally, you should mix it immediately before using it, and store for no more than an hour.

You can scale down but it will require either pulling off the dropper part from the bottles each time in order to pipet the required quantity (for example, 10 ul of chromogen and 500 ul of substrate). It will be better to transfer the contents of each reagent to a new container (for example, an Eppendorf tube wrapped in foil to protect from light for the chromogen and a 15ml tube for the substrate).

I can confirm the Protein Block is a PBS solution with 0.5% Casein and 0.5% BSA.

I hope this information will be helpful for your customer.
Have a great day!

Thank you for getting in touch.

When using frozen sections, you can use a general frozen section staining protocol. I would amend the protocol booklet protocol as follows:

1. Fix the sections (for example 4% PFA for 10 minutes or ice cold acetone for 5 minutes). Fixation will need to be individually optimized.

2. Add enough drops of Hydrogen Peroxide Block to cover the sections. Incubate for 10 minutes. Wash 2 times in buffer.

3.  Wash 3 times in buffer.

4. Apply Protein Block (if required) and incubate for 10 minutes at room temperature to block nonspecific background staining. Wash 1 time in buffer.

5. Apply primary antibody and incubate according to manufacturer’s protocol.

6. Wash 4 times in buffer. Apply Biotinylated Goat Anti-Polyvalent and incubate for 10 minutes at room temperature. Wash 4 times in buffer.

7. Apply Streptavidin Peroxidase and incubate for 10 minutes at room temperature.

8. Rinse 4 times in buffer. Add 30 μl (1 drop) DAB Chromogen to1.5 ml (50 drops) of DAB Substrate, mix by swirling and apply to tissue. Incubate for 1-10 minutes. Rinse 4 times in buffer.

9. Apply counterstain according to manufacturer’s instructions.

10. Dehydrate if required and coverslip.

We have further information on IHC protocols on the following web page which I hope you will find helpful:

https://www.abcam.com/tag/ihc%20protocols

Which primary antibody are you using? This kit can be used with rabbit primary antibodies. The antibody can be diluted in PBS 0.2% Tween and include the protein block,

Wash buffer should be PBS 0.2% Tween

I am happy to answer any further questions you have.

The lab sent me the following explanations:

The protocol for ab64261 reflects the sequence we follow in house. 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.

The Protein Block step can be used as necessary for up to 10 min to help decrease non-specific background. All incubation times were optimized during the development of this product.

I am sorry that I was unavailable when you called but am glad to hear from you. We would not suggest using 5% BSA instead of the protein block included with the kit. These blocking solutions are formulated to give the best result with the kit. I feel that as you have continued to experience problem with the antibody that it may be that the vial or lot which you have received is faulty. I believe the lot which you have is xxx. We currently have a new lot available which I would be happy to send to you. Alternatively under our Abpromise guarantee I can credit/refund this product for you. https://www.abcam.com/index.html?pageconfig=abpromise I hope that this information is helpful. Please let me know if you have any questions or there are other ways that Abcam may help you meet your research goals.

Thank you for contacting us. You will need an antigen retrieval step. Customers have reported success with a few different protocols but I recommend a heat mediated retrieval using a 10mM citrate buffer, pH 6.0. This should be applied to the sections before the peroxidase blocking step. After deparaffinization, simply heat the citrate buffer to boiling, immerse the re-hydrated slides in it, and maintain the buffer boiling, at 95-100C, for 20 minutes. Allow to cool for another 20 minutes and continue with the protocol. Here is a link to our IHC guide, which includes a section about antigen retrieval.

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

The thickness of the sections may be an issue since the antibody recognizes an intracellular domain of CD31. If the diameters of the cells in the sections are less than 50 microns, then some will likely be intact, and the membranes of those cells needs to be permeabilized to allow the antibody access to the cytoplasmic domain of CD31. Include some detergent in the blocking buffer, for instance 0.1% Triton X-100, to permeabilize the cells.

The Invitrogen mounting medium is aqueous, which is required for AEC. AEC dissolves in organic mounting media, resulting in no stain. The mounting medium is also compatible with the DAB in the ab24621 kit.

I hope this helps. Please let me know if you have any questions.