min6 cells immunofourescence staining - staining and culture problem (Jan/10/2011 )

Hi everybody,
I have just begun to learn min6 cells staining but i couldn'i take any results, i can not see green flourescence, primary ab-secondary ab is compatible nad has no problem, give me please advise?

It's really hard to give you advice and suggest where you can potentially change your protocol with so little information. Perhaps if you post what it is you are doing we would be able to help a bit more. For example, how are you fixing your cells? What is your staining protocol? Are you certain that the antibody you are using works in IF analysis? Are you certain that the protein of interest is expressed in min6 cells (these are very specialized cells). Are you trying to detect an endogenous protein or are you overexpressing first? Do you have a positive control so that you know your secondary antibody is good? See what I mean? There are so many potential areas of problems that we can't help you pinpoint where your specific experiment is going wrong without more information on your part. Sorry!!

Its been a while but when I did this I don't think I used a fixative... if i did i can't remember what it was. I cultured my cells in small dishes with square glass coverslips in the bottom. When I was ready to stain I washed the cells VERY GENTLY with PBS 2X. Then applied my primary antibody for 30 minutes. washed again very gently with PBS 3X. Then applied my secondary antibody in a similar way.

When my slides are ready I put a drop of DAPI on the slide and then put it onto a glass slide. You can use clear nail polish to seal the edges of your slides. If you store your slides at -20C. They should be alright for a while.

So so Thank you for your reply, i will try as you said, because my reagents work perfect and i just follow the protocol..i hope it will work at this time..
Best wishes...
Duygu

Duygu on Mon Jan 24 19:34:48 2011 said:

btw, make sure u work in very dim light bc the fluorescent proteins will be bleached out by light. i usually worked ina room w/ the light off and cracked the door enough so I could see what i was doing.