I am doing IHC (primary ab p-Akt) on mouse cardiac tissue. At the final step of Vectastain ABC followed by DAB chromogen, I could visualise the brown colour changes, but colours disappear and pretty much washed away as soon as counterstaining and rehydration. Does anyone have any idea why that may be the case? Would expired kits have anything to do with it?
(i tried two different counterstaining - methylgreen, and haemotoxylin - both of which did the same thing)
I never had this kind of problem and normally DAB isn't solvable in water. So the expired kits is probably the answer.
DAB chromogen is able to survive after ethanol and xylene treatment. Usually, when the intensity of reaction is very low, the brown color of DAB is completely masked by haematoxylin. You could try to dry the slides before mounting and check.
I have run into issues with DAB reactions when using Vectastain ABC that was past its expiration date by as little as 1 month. I would use a fresh kit without any counter-staining to check if this is the problem.
sorry. You could try drying the slides before deshydratation and afterward mount them in order to verify the potential effect of organic solvents in DAB precipitate.
Have you tried using metal enhancer with the DAB? It changes the staining color to purple/black instead of brown. Maybe your counterstain is masking the DAB.
Thanks all, this forum was tremendously helpful. I am ordering new Vectastain ABC kit and Vectastain DAB substrate kit, so finger crossed!
I am a 'newbie' when it comes to lab work. When I was counterstaining with methyl green, I have discovered two things - the methyl green in my lab is in fact ethyl green with methyl violet contamination - so I have to chloroform seperate it. Secondly, little did I know water and ETOH removed most of the stain like a flash! Any advice in using this counterstaining protocol? (I prefer this over haemotoxylin as I found the blue is quite close to the brown/black from DAB/Nickel).
Odd - the DAB reaction product is NOT water soluble and is about the most stable chromogen complex available. It can be enhanced for more contrast using a nickel, or nickel-cobalt reaction that renders it black rather than brown, but neither should wash out with a counterstain. A more sensitive chromogen, tetramethyl benzidine (TMB - Mesulum, 1982) is notoriously alcohol labile and excessive dehydration reduces the signal. I have never heard of this problem with DAB. It's possible something was wrong with the reagent. I used ABC kits but I always used DAB from Sigma and titrated the oxidation with hydrogen peroxide rather than using a kit for the reaction.
We never had this problem with DAB, so I believe it could have to do with expired kits, or with storage (too warm or too cold). I recommens to look for another chromogen which is called VIP (very intense purple). It can be used like DAB, is not carcinogenic (says the company), and there is a ready for use methyl green stain which intensifies the purple.
We've found that dehydrating NovaRED with ethanol washes away the chromogen, so something I've recently started doing with good results is dehydrating on a heat block at about 55 C for ten minutes then 2x2 min xylene dips and coverslipping. You could try that with methyl green
Iyas - that is an interesting observation -- I've never experienced ethanol removal of Nova Red. Nova Red is supposed to last 50+ years in stained sections - and has always been very stable through 1000's of slides I've dehydrated through graded ethanol solutions then into xylene. I'm surprised to hear that about Nova Red (which has long been felt to be superior to DAB Ni/Co). I will now be more careful about ethanol and Nova Red. Good to know that this could cause loss of Nova Red signal.
What kind of sections did you use when you observed loss of Nova Red by ethanol?
I'll get back to you on more specifics later when I'm in lab, but essentially what we found was that the 80% ethanol dip was turning pink over time. At first we assumed it was due to the container of Eosin next to it creating the illusion of a pink ethanol color, but when I took the liquid out and dumped it into another container, the color persisted so I made the conclusion that it was due to NovaRed coming off the slides. The tissue we were staining was mouse tissue in relatively thin sections (10 microns?)
When I was doing research on the topic there were very few people experiencing the same problem as us- for the most part everyone echoed your statement regarding NovaRed's stability in ethanol.
Iyas,
I have noticed the pink/reddish color too, in our 70% ethanol...
Hopefully it is just excess Nova Red coming off... and not the specifically-precipitated target Nova Red (but who knows). It is good to be aware of this - I very much appreciate your observation.
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