Just in case anyone else cares to know...
Hey Ham Fan,
First of all, can you answer some questions for me:
1. What tissue or cells are you staining (I'm assuming tissue staining via ABC (Avidin-Biotin Complex) substrate since you are using Vector Red)?
2. Frozen or paraffin sections?
3. What are you using for blocker?
Problem 1: What do you mean by background? When adding your Vector Red (BTW I've used Vector NovaRED...I think you are referring to the same substrate) I usually let it stain for 20 minutes. The binding affinity of these color stains are not as good as DAB. Regardless, there will always be some background staining. I think that the problem may be that you are using too high of a concentration of primary antibody. By oversaturating the primary antibody, you are getting non-specific binding and ultimately getting staining on other areas which appears to be background staining. I suggest you do 2 things. First you should get 1 slide and skip blocking, primary, secondary, and ABC. Just fix, perform antigen retrieval (if you are using paraffin sections), wash through PBS or TBS and add the Vector Red. View under a microscope (by eye, you will almost always see a red hue to the tumor...this means nothing) if you see positive staining, you have endogenous peroxidases and will need to block them. The best way to do this is to add 0.3% H2O2 before your blocking step. If this is the case let me know...I will give you more advice. The second thing I suggest is that you perform a titration analysis, which is a must for good IHC staining when using a new antibody. If you are using anti-serum, I would suggest 1:10, 1:50, 1:100, 1:200 dilutions of the primary antibody. Run them all together making sure you add a different dilution of the antisera to each of your slides and make sure they don't mix. If using anti-IgG, try dilutions of .5 ug/mL, 1 ug/mL, 5 ug/mL, and 10 ug/mL. I usually have best success with 1-2 ug/mL. If you are using a self-generated antibody, let me know...I have more advice for this.
Problem 2: Again, I've only used Vector NovaRED before, but I think we're referring to the same product. From what I remember, after making the solution, it is clear. However when you add it to peroxidases it will turn red. The left over solution is still clear.
I have no idea what you are using the Tris for? Are you making some of the solutions on your own? I buy most of my substrates from Vector Labs and make my own blocking solutions and generate my own polyclonal antibodies by immunizing rabbits...fun stuff.
Sorry this was so long, feel free to PM me. It took me 4 years to perfect IHC staining and I had to learn alot of it on my own which really sucks. 😡 Good luck!
