How to fixate cells onto glass microscope slide - (Nov/13/2008 )
Hi
I would like to fixate my cells culture in microscope slides but i tried last week with no sucess.
Any good advice???
Thank you in advance!
Reis V.P.
Are you trying to grow them on the slide or just trying to fix a mounted cell clot or already grown cells?
If you are trying to grow them, put coverslips in the wells of a 24 well plate and add your cells as you would normally. You may need to gelatin, poly-L-lysine or collagen coat the coverslips to get the cells to grow. Otherwise BD and a couple of other companies sell cell culture slides with chambers on them expressly for this purpose.
Cells that are attached to a slide or coverslip should be washed a couple of times with ice-cold PBS and then fixed for 15 min with 2% paraformaldehyde, then washed 4-5 times for 5 min each with cold PBS. For the last wash leave overnight at 4 degrees in PBS, this will remove any remaining free aldehydes left over from the fixing and thereby reduce your background staining.
I would like to fixate my cells culture in microscope slides but i tried last week with no sucess.
Any good advice???
Thank you in advance!
Reis V.P.
First, what type of glass are you using? What cell line? As mentioned, it's highly suggested you look into coated coverglass. Many cell types can't adhere well onto plain glass. You have many options including collagen, poly-L or poly-D lysine, fibrogen or combinations of these. There are others but these are the most common. You can buy the glass already coated (I agree that BD Bioisciences is a good company) or there are many protocols for coating your own glass which is much cheaper but generally less reliable. This coating gives an extracellular protein base for the cells to grab onto.
Next, what type of fixation are you needing? Paraformaldehyde (I do 3.7% for 15-20mins) is the best I've seen for maintaining adherence but can be bad for visualizing specific intracellular substrates such as microtubules and centrosomes. Generally these structures are better seen with -20degree methanol fixation. There are actually many different types of fixation good for maintaining specific cellular integrity but each comes with it's own pros and cons.
Did you look at your cells before fixation and see many cells that at least appeared to be adherent to the glass? By any chance are you arresting the cells in mitosis? Mitotic cells easily wash away and take great care to maintain through fixation. Hope this was of some help.