As far as DAB and AEC are both developed by peroxidase, I think it is difficult to combine both in one protocol. There would have been a step between the single-stains, that denaturate the first added peroxidase - or block it again like the endogenous peroxidase?

Thanks for your answer!

I'm using Biotinylated 2°Ab and then HRP-avidin-Tyramide followed by DAB revelation.

I was thinking about using a AP-2°Ab and then reveal it by AEC. 

Does that make sense? It is just that I don't have any protocol for that and there are many different products on the market (DAKO, Vectorlab..)

Dear Christophe combining DAB and AP is good. we use this technique for double staining. However, we also use DAKO Envission double staining kit it results even better. The DAKO protocol is very robust and easy to use. If you have have an automated staining station then it can be optimized there also.

Dear Shibabrata,

thanks a lot for your answer.

Could send me a protocol then for the double staining you use?

Unfortunately, I'm doing all staining by myself..

Bests,

Dear Cisarovsky, I do not have a protocol. However I think that it is possible to perform a double immunohistochemical staining using DAB and AEC. But as the AEC chromogen is soluble in organic solutions you must use an aqueous mounting medium such as Ultramount (Dako. NO.:S1964). I performed this technique in a postgraduate course and I had good results.

Best regards,

Thanks for your help!

The following is the protocol we use for the duoflex kit  from DAKO. It gives excellent results.

For double staining, sections were deparaffinised and dual staining was performed as per manufacturer’s protocol.

1. Target retrieval was done using EnVision™ FLEX Target Retrieval Solution (1x working solution) and incubated for 30 minutes at 97 °C.

2. Then the sections are washed in room temperature with EnVision™ FLEX Wash Buffer.

3.Then DuoFLEX Dual Endogenous Enzyme Block was added and incubated.

4. After endogenous enzyme block, the sections were incubated with primary mouse anti human CD4 and rabbit antihuman CD8 antibod for 1 hour.

5. The sections were washed ones with the wash buffer.

6. Followed by incubation with the EnVision™ DuoFLEX /HRP reagent and then EnVision™ DuoFLEX /AP for 30 mins.

7. The reaction was visualized by EnVision™ DuoFLEX DAB Chromogen for mouse antibody and EnVision™ DuoFLEX Permanent Red for rabbit antibody for 10 mins. Keep looking under the microscope for the correct development.

8. The sections were then counterstained with hematoxilyn for 20 secs (depending on strength)

9. Kept under tap water for 5 mins.

10. Kept in distilled water.and then air dried.

11. Cleared in xylene by a plunge and mounted with aquas mount.

Christophe, do I understand you right? You want to combine Alkaline Phosphatase (AP) as enzyme and Aminoethylcarbazol (AEC) as chromogen?

AP needs a reagens, that contains a phosphate-group, as substrate. This phosphate-group is cleaved. The rest of the molecule combines with another molecule to form a chromogenic substance.

AEC (like DAB) is used with peroxidase as enzyme. Peroxidase cleaves the H2O2. The produced hydrogen is added to AEC (or DAB). This reduction forms a chromogenic substance.

Thank you Shibabrata Mukherjee! So you don't use any 2°Ab? Are your 1°Ab already conjugated?

@Gudrun Lang: Yes I saw many people using this double staining techniques but I was looking for the products I need to buy and a protocol to use, as I already have one that works well but only for DAB. Does it sound impossible to you?

I never heard, that someone used alkaline phosphatase for developing AEC. For double-staining: first IHC-part with peroxidase and DAB, second IHC-part with alkaline phosphatase and eg. BCIP-NBT or permanent red http://www.dako.com/at/ar38/p224598/prod_products.ht