Weird Immunofluorescence - (Feb/18/2015 )

Hi All

I am very amateur in working on IF and microscopes. I recently did an IF with HEK293 cells that I knocked down a gene of interest and did an IF with its antibody to "optimize" the use of this antibody, as well as showing its specificity.

I used a serial dilution (1:200, 1:500, 1:1000, 1:1500, 1:2000, 1:5000) for the primary antibody and kept the rest the same (blocked with 5% BSA 1 hour, Secondary antibody 1 hour RT)

One thing that I noticed under the microscope is that, I can see dotty red staining in all over the cells even in very high dilutions (Albeit weaker). When I took photos with the camera to compare the photos from knockdown and control, I notice something very weird. Most of the photos looks like the attached photo again dotty red staining. When I used the same exposure time to look at knockdown samples, I saw nothing! Like absolutely black, which can not be right.

But, somehow I managed to get couple of better photos, did not happen more than couple of times and only on specific fields. I do not know what I did. They look like the second photo attached. You can see the red staining (I uploaded only c3) is clearly more in nucleus which makes sense since this protein is nuclear. and this gets reduced on KD sample when the same exposure time was used.

Sorry, I am so dumb in microscopes please help me figure out what I did wrong. I used axiovision software and just tried to keep the Exposure time and % pixel integrity similar when taking different photos. however, it is very field dependent and one field gives a better photo than the others. Also I tried the linear and best fit options, but did not understand what I was doing...Also the camera seemed like it cannot find a proper exposure time automatically. Like it would set it on 2 ms and when I took the photo it would give me a black field so I had to adjust it manually...

Thanks all for your help and comments. This is so frustrating :(

Dotty staining could be a function of either a non-specific (secondary probably) binding of an antibody or too high an exposure. There is also the possibility that you have a low level bacterial contamination of the cells, which could cause non-specific binding. Uneven staining across the slide could be a result of not enough fluid covering the cells when staining or the coverslips drying out.