I miss the blocking of endogenous biotin in your protocol.

http://www.ihcworld.com/_technical_tips/biotin_tips.htm

http://www.rndsystems.com/resources/images/6903.pdf

http://www.dako.com/ihc-guidebook-background-chapter15.pdf

http://web.bf.uni-lj.si/bi/mikroskopija/pdf/Technical-IHC.pdf

please tell the community the complete IHC protocol with all blocking- and incubation-steps.

The picture is not really clear, anyway I've used another NG2 antibody and never had any problem. Why don't you use AP for pericytes? for IH works much better.

Ornella

Non-specific background staining can be due to a number of factors. Assuming that you are correctly doing antigen retrieval. as well as peroxidase and non-specific protein blocking steps prior to your incubation, the first thing you should do is dilute down the antibody progressively, until you have no more background staining. If your target cell compartment still stains positive at a concentration that has no background staining, then you have found a correct working dilution. If by diluting down you lose both the non-specific background and the expected positive staining, then your antibody is probably not specific enough (or at all), and you should probably find another. By the way, as a rule I stay away from all Santa Cruz products.

Gudrun Lang: The complete protocol is attached.

Jean-Martin Lapointe: I tried diluting the antibody, and then I progressively loose any staining. I get the staining at fairily high concentration 1:20. I am more worried for the nucelear staining even in negative controls, I read somewhere that nucelar enzymes can develop DAB.

Ornella Capellari: Which AB did you use? I used DAB because it seemed easy with the kit to start with. Is there also a kit for AP?

Well, the c-Jun from Santa Cruz is ok and also the beta-actin :)

There should be no difference between POX an ALP staining. ALP substrates give more "linear" stain, which might be beneficial in spindled structures.

If you lose your stain in progressive steps and if the dilution is 10-15 times, this is ok. But if you lose your stain, like two times, between 1:100 and 1:200 for example, I will doubt the antibody specificity

Best,

Ivan

Hi Olga,

what you describe, staining at a high concentration (1:20 is quite high) and then losing all staining with dilution, is suggestive of a non-specific reaction. If you can't get good staining in your target area without having background staining in areas that couldn't possibly have the marker, then that antibody will be of little use.

For the nuclear marking: you mentioned that you are getting "black" staining, in both test slides and negatives. It's difficult to be sure without seeing photos, but I think what you are seeing is an artifact that has nothing to do with the IHC. I have seen it before, and I don't have an exact cause for it, but it looks a little bit like gas or air in the nucleus, which refracts light and gives nuclei this dark to black appearance. DAB, even with intense staining, always looks brown, not black.Look at the nuclei at high magnification, and see if you get a suggestion of bubble appearance. I think it may have to do with a problem during processing or slide mounting.

I miss the blocking of endogenous biotin in your protocol.

http://www.ihcworld.com/_technical_tips/biotin_tips.htm

http://www.rndsystems.com/resources/images/6903.pdf

http://www.dako.com/ihc-guidebook-background-chapter15.pdf

http://web.bf.uni-lj.si/bi/mikroskopija/pdf/Technical-IHC.pdf

correct dilution is the main cause of un -specific binding. i think u need to work with the optimized dilution for your experiment. a rule to stic is dilute down the antibody progressively,

you should check some steps again;

1- dilution of AB by making serial dilution then select the best one without non sp. and good positivity.

2- keep slides witted during staining steps.

3- use fresh DAB.

Good luck

I would do the following:

1. Get a good antibody from Abcam (LHM2-cat # ab83508, 100.0 ug/$392.00) or Novus Biologicals. I looked at their ( Abcam) images and it does not look the antibody stained nuclear areas.

2. Try using Vector laboratories kit  for following the ABC technique. In case you run out the secondary antibody, you can always buy them separately. Also probably there are lots of dead cells and they stain quite easily with anything.

3. Talk to the tech service of the company. They are really good in giving you right directions about dilution, non-specific blockers etc.

4. Dilution of the primary and secondary antibody matters a lot.

5. Prepare fresh DAB solution.

Good luck.

 Hi,

1) No more SC

2) To reduce inespecific label in the nucleus try Streptavin Kit instead avidin. There are a protein electric charge problem. For detailed information go to Bioconjugates Techniques T. Hermanson

It looks to me as if you have air on the section due to drying out while mounting, as mentioned above by Jean-Martin.  I have seen the same thing when people allow the xylene to evaporate before mounting. Be sure the slide is still wet when you put the mounting medium on. Always put medium on a wet slide and not on the coverslip. Good luck anyway!

HI Olga, I Have checked your protocol. There a some things you can change, but they are not essential for your unspecific staining in your negative control. Try a higher normal serumcon-centration during your blocking-step. I recommand min 10% normal serum. And if this does not work, change your detection system. Use the ABC-System like Ranu Pal recommands.  And do the over night incubation of the primary ab under room temperature. Advantage: you can use higher delutions and this helps for better titration of the optimal AB-concentration. Good luck.

Hi

1) no more SC

2) use a Streptavidin kit instead avidin. Read the related chapter in Bioconjugate Techniques by Greg T. Hermanson. You could see a problem with electric charge so that yields inespecific nuclear background.

Hi Olga.

Looking at the pictures you posted I think something is wrong with the microscope settings you used. You should take a look at your slides without any phase contrast filters in the light path and/or with opened condenser lens, because these might give you false results. Could you please post some pictures taken without PH filters and with wide opened condenser?

Greetings