I am writing this for people who have used the Kit described below and have thus omitted most of the procedural information on the understanding that all steps not commented on where followed as in the instructions without deviation. To anyone who has not used the kits the protocols are the same for all Ab, as are the reagents, except for the primary Ab. Having just completed our first immunos using the "Kwik Kits" from Thermo Shandon. (We where given everything as a sample, including the pre-diluted Ab)
We ran the Ab, one with an overnight incubation(HMB45) and one using the protocols 30 minutes for incubation(S100).
Both of the protocols worked, the differences being the primary Ab, the length and temperature of incubation and the time from which the chromogen was made up. For the S100 the Chromogen solution was made up 20 minutes before being used and the chromogen solution was clear, with only the slightest hint of red when applied to the slide with the S100 tissue and control.
For the HMB45 the Chromogen (Fast Red) solution had been sitting for 14 hours in the fridge (temp 5 deg. C) in the dark ( the instructions give the maximum useful storage time to be 24hours) the solution was now very clearly red).
The end reaction of both was clearly positive with no background staining, I was very pleased by this, but the staining on the HMB45 was much more pronounced, where as the staining on the S100 was weaker, more like a pastel shade of red.
Between the two methods there are two many variables to draw any conclusions on why the staining varied so greatly.
My questions are these, has anyone using these kits found the quality of the staining to be any better or any worse with the chromogen having left it sit to "ripen"?
Or am I looking at the advantage of an overnight incubation?
If you have investigated these alternatives I would be grateful for your input.
I have contacted my representative concerning these matters and I am still posing these questions.
Finally I have been using an aqueous mountant, I am finding it difficult to stop lots of bubbles from forming, what techniques are people using?