Hello,
I have a new batch of Hardset Vectashield that I forgot to put in the fridge one day and it spent a night on the table. Now I used it to mount/counterstain some cultures and the result looks very bad: DAPI fluorescence is extremely dim and I can see plenty of fibers and garbage on top of cell layer. I checked all the solutions for dust under the microscope, and Vectashield that I used had some fibers in it. When I compared it with a drop from completely new Vectashield bottle that has been never used yet and was stored in the fridge, the brand-new one also had some fibers but much less of them.
I think I also have problems with cell fixation because cell layer looks rugged and "gunky", so maybe the dust in Vectashield is not the main culprit here.
Do you have any experience with Vectashield gone bad? Sorry for not giving you photos of my problem, I can't take them right now.
Are these fibers or air bubbles? The hardset Vectashield should be stored tightly capped in the cold...I would not use the one that has been left out, especially if it was not capped properly!
Air pockets can form as the Vectashield sets. Could this be why your cells look gunky? We usually use the non hardset one, and then seal the coverslips with nail polish. Have no problems.
Thank you for your answer. I'm pretty sure these garbage particles are not air bubbles because they're not round. I would say that it looks like sand under magnifying glass, and my colleague said "it looks like oatmeal" (again, sorry for not providing a photo). We decided that the surface of the overgrown culture was not flat and I used too little Vectashield, so it solidified as a thin film on the uneven surface of these cell (or debris) piles, and now the light scatters a lot making cells hard to see. Maybe it would be better it I used enough Vectashield to cover the dish completely so it would solidify with a flat surface like a puddle. What do you think about it?
The exposed bottle was on RT but was tightly closed all the time.
Byron, I did this and I described it above =)
Since this day I have tried a few other mounting media and in my experience VectaShield was just the worst. Even fresh and clean Vectashield is not fully transparent.
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