Avoid drying out of coverslips during immunofluorescence - (May/27/2015 )

Dear BioForum users,

 

I have recently started performing immunofluorescence on fixed mammalian cells.

 

Although the steps are pretty straightforward, the coverslips carrying the cells would always tend to dry out between the different incubations.

 

As a result the antibody solutions won't spread well and evenly on the cell layer. I am afraid that this problem generates artificial differences in the staining intensity besides the high background.

 

I always try to leave some small amount of fluid (eg PBS) from the previous steps on the coverslips but on the other hand I am afraid that this might overdilute my antibody and distort the initial concentration.

 

Could anyone please suggest ways to avoid drying of the cells between the different IF steps? Any help would be really appreciated.

 

Thank you!

do you incubate in a humidified atmosphere? you can set up a humidity box in which to perform the incubations.

Thanks mdfenko.

 

I actually do what you suggested. The little detail I might be missing is the little time that elapses between aspirating the PBS (or whatever washing solution) and pippeting the primary/secondary antibody on the coverslip.

 

I still cannot find a perfect balance between not overaspirating the washing solution but also not leaving too much on it to avoid diluting the antibody.

 

I guess it's ok if it is like a minute on average that part of the coverslip is not covered with a solution but I cannot be 100% sure how fast the cells dry out.

how do you contain the solution on the coverslip? we surround the area with a "pap" pen.

 

if you work quickly enough then the sample shouldn't dry out during the interval between removal of the wash solution and application of the antibody or developing solution.

That's exactly the point.

 

I try to be as quick as possible but sometimes the antibody or whatever solution won't just spread very well on couple of the coverslips. Because of this I have to spend some time redistributing the solution in order to cover the most part of the coverslip (if it is only at the center at the beginning). I guess this procedure does not take me more than 2-3 minutes (for six coverslips in total) but I don't know if during this time there can be some parts of one or two of the coverslips which dry out.

 

We always tell the technician to only image the cells in the center of the coverslip however even so I often have variability in the signal intensity between samples of the same group and this is what makes me sceptical. 

 

Thanks again for your valuable comments!

do you handle one coverslip at a time or do you remove wash from all then add antibody to all?

 

there should not be significant drying of the cells if you just remove wash and then immediately add the next solution.

I actually remove wash and add antibody to all. 

 

In my hands it is impossible to do one at a time because to effectively remove wash from each one I have to tilt the plate and this causes the antibody solution already being on other coverslips to spill over.

 

Do you have any suggestion as to how I could avoid this?

 

Thanks again for your help, I really appreciate it!

you could remove the solutions with gentle suction (stay away from the cells when doing this, take the solution from the corner), no need to tilt.

 

if you have a bunch of coverslips then it will take too long to add the solution without drying.

Or simply don't worry about removing the buffer completely. Leave some buffer behind between washes. Dilution will take care of the rest. Sounds like you are working in a very dry environment. The humidity in my lab is 40-50% and I've never encountered this problem so far.