NBT solution in X-Gal staining - (Nov/08/2012 )

Hi,

I've been transfecting some cells with LacZ expressing constructs, fixing them and then staining with X-Gal using Ferri and Ferrocyanide to make them blue and see where my reporter is expressed. This protocol works really well but I only get a few stained cells because the transfection isn't very efficient and my construct is low expression.

My supervisor suggested that I try using NBT instead of the cyanides, because it is apparently more sensitive therefore more cells will show up. The protocol that she has given me from an old paper said to dissolve the NBT powder in 100% ethanol at 100mg/ml and use 4ul of this per mL of staining solution.

I tried this and straight away I could see there was a problem - the powder was no where near dissolved. I did some research and found that apparently NBT is only soluble in ethanol at 5mg/ml, so I diluted the solution down to 5mg/ml in 100% ethanol and used 80ul/ml.

The powder was 99.9% dissolved so I added it to a solution of PBS and 5mg/ml of X-Gal (dissolved in Dimethyl Formamide). I added it to my fixed cells and put them in the dark to incubate at 37 degrees C for a few hours, like I normally would with the X-Gal and cyanides.

After 2 hours I checked to see if the stain was coming up - all I could see was a cloudy yellow solution with precipitate and cells floating around in it. None of my cells stained purple like they were meant to. They all looked really unhappy.

Do anyone have any ideas of how I could fix the staining protocol? Is it just that my ratios of X-gal to NBT are off, or is it because I dissolved the NBT in ethanol and not DMSO/DMF, which I know is how most people do it (to do this I would have to order more NBT powder, which I don't want to do unless I have to). Would the DMSO/DMF do anything to my cells? Could it be that I made the stain in PBS and not something else like water?

Any help is appreciated!

Kristen

Ethanol, DMF and DMSO are all toxic to the cells - ethanol will probably be tolerated to the highest level but you had about 8% in your final concentration - this is toxic for many yeasts, let alone eukaryotic cells.