Dear Yuwei,

From what I gather from your description "only a few dot points" is that you may not be using your confocal microscope effectively. You should be seeing at least some labelled nuclei with DAPI, you cant go wrong with DAPI. Usually you should also see some background at higher power. That kind of a few dot points imaging happens under extreme conditions such as the right laser source is not "turned on". Make sure that you get help from someone who regularly uses the confocal setup and he/she helps you set up the pathways for the right excitation and emission wavelengths. 

best wishes,

Refik

(1) according to my personal experience, PE is not a good choice for confocal even it is quite bright and widely used in FACS, it is very easy to be bleached in Confocal, that's may be one possible reason for your only dots, you need to change your streptavidin conjugated fluorochrome, such as alexa fluro are most good.

(2) It is very useful to check your staining efficiency by FACS before you go to Confocal. If the brightness of your target protein is less than 1 log comparing to your negative control, you may need to struggle hard to get enough signal from your slides.

(3) play with and optimized confocal machine is helpful as well.

Hi all,

Thanks for your advices.

I have attached my results. Actually the staff in microscopy unit help me set the machine, and we have tried the higher power laser. I attached the setting information of the result: the excitation wave of PE is set at 561nm with 85 % (20 % and 60 % tried before), and emission wave of PE is set from 575 nm to 630 nm. PE is easy to be bleached indeed, and I tried to use DyLight 488, the excitation wave of 488 is set at 488nm with 20 %, and emission wave of 488 is set from 505 nm to 561 nm. As FITC was used in sorting cells, I do not want to use 488. Is APC good for confocal microscopy?

In FACS result, the CTLA-4 is not strong enough, only 1 log bigger than negative control. I was recommended to increase antibodies concentration and improve permeabilizing steps to enhance signal.

APC is good for confocal microscopy, I often use it. But most important, I think you should find ways to enhance signal. According to my experience, 2 log bigger than negative control is needed.

I would try, as Shiquiu suggested, to check by FACS that the staining worked before going to the confocal and use confocal friendly dyes. If you do not like the 488 use 568 or 594 instead (Alexa, Atto or Dylight). Optimising the settings (LASER & Filters, high NA lenses) it is also very important. Also, if possible, use a LSM with the new avalanche or hybrid detectors instead of PMT.

In addition, if the signal it is very weak you can try to open the pinhole a bit. In a LSM usually it is set to 1 AU (that gives you the best Z-resolution) but if you open it, the Z-resolution will be slighly worse and the signal to noise ratio a lot better. This is because in a LSM the Z-resolution improves linearly when you close the pinhole but the amount of light that reach the detector decrease exponentially. You can open it up to 2 or even 3 and still get a fairly decent confocal section of very dim structures.

Dear Yuwei:

(1) BD Cytofix/Cytoperm should be ok for fix and Perm, but I prefer my own fixative and Perm. Cold absolute Methanol fix cells dropwise and fix on ice less than 2 hours will expose most epitope, which is better than 2% PFA even PFA maintain the cell morphology better than methanol. PBS+20%FCS+0.1%Triton X-100 is my homemade favourite Perm, which worked well in all my transcription factors.  

(2) I normally keep laser power frequency less than 20%, most flurochromes will totally bleached if you use high power. To collect enough signal, enlarge pinhole a bit may be helpful.

(3) The CLTA4 expression level looks fine, we will get your picture soon.

I would start very early in the procedure. The time of incubation with your permeabilizing reagent makes a HUGE difference to your staining. I typically do a 4% PFA fixation followed by either 2min in PBS/0.2% TX-100 or use a saponin based permeablization method that is excellent for vesicular structures. 

Methanol is excellent for nuclear proteins, but if used for more than 2 min, then you lose a lot of cytosolic proteins.

What is your protocol for fixing/permabization?  

Hi everyone,

Thanks for advice. The high power laser did bleached the signal, and I will try to enlarge pinhole further next time to collect more signal. I think we did use hybrid detector before, and it still very dim signal.

I tried to improve my permeabilizing step this weekend. I used BD cytofix/cytoperm kit and BD FoxP3 buffer to compare the permeabilizing step. Both of them contain PFA and saponin and made no difference based on my FACS results. But the temperature made a difference. I usually stained cells on ice, but after following the advice on temperature from manual, the CTLA-4 positive cells in FACS result increased a half log. I hope that could be helpful.

And I was recommended to increase the antibodies concentration further.