Posted 07 February 2012 - 08:13 PM
In the beginning, I am very grateful for any one who will read my topic.
Secondly, I want to buy some culture wares for visualization of my cells under fluorescence or phase contrast microscope.
what I found due to reading some books and online:
*There are special type of culture slide such as
*It is untreated.
I should treat the surface with Poly L lysine or collagenase as I am working with adherent cells
but I cant know the difference between these reagent and which on is the best.
* After culturing, Does the step of fixation is necessary, or adhesion to surface of my slide is enough.
* Shall I use the ordinary glass cover on my slide or use these precoated glass covers or precoat them by myself.
finally, does these steps differe if I will use a laser scanning confocal microscope?? Does any one has experience with it and ready to share??
At the end I am deeply indebted for those who will share their thoughts, experience with me.
Posted 07 February 2012 - 09:45 PM
Posted 08 February 2012 - 03:57 PM
I typically use 10-13 mm diameter coverslips in wells of a 24 well plate (they can't flip over in the wells, when you are getting them out), and square or rectangular coverslips work well in 6 well plates.
These procedures do not change for confocal.
Posted 08 February 2012 - 05:01 PM
I am very grateful for your precious comments.
You mean that I dont need to added any thing such as poly l lysine, or collagenase, or fibrocetin to ensure the attachment of my cells into glass slide (even culture glass slide are not treated, unless you buy it treated.
Secondly, so I can use sterile cover slip and drop it in my culture dish, then take it out, invert it, and put it in slide and watch my cells under microscope, is that what you meant doxirubicin,
I dont use ABs, so I guss fixation and permeabilization step is not required at least for now.
Posted 09 February 2012 - 09:01 AM
I imagine you probably will just want to fix the cells to observe your protein, however, so that you can make "permanent" slides. To do this, you can sterilize coverslips by soaking them in 70% ethanol, then place them into 6-well dishes. Either let the ethanol dry or wash the wells one time with sterile PBS. Then trypsinize your cells and plate them in normal medium on top of the slides. After the cells are growing fine (24 hours), you can then wash away the medium, wash cells with PBS, and then fix/permeabilize the cells. You can probably get away with just adding -20C Methanol to the cells and letting them sit for at least 30 minutes. Next, remove the Methanol and wash with PBS. You can then pick up the coverslips with forceps and flip them over onto a normal slide. You will likely want to put a mounting medium (anti-fade reagent) in between the two pieces of glass and let the reagent dry at room temperature in the dark for a few hours. Then seal around the edges of your coverslips with clear nail polish, and voila, you're ready for confocal.
Posted 09 February 2012 - 10:32 AM