I need to observe intracellular protein in my suspension cell line through fluorescence microscopy, but I am not able to get good staining and proper method for attachment of cells to glass slide...we are not having cytospin at our facility...so anyone tell me a method for attachment of suspension cells to a glass slide?
You can fix your cells in suspension. Next, wash the cells and resuspend them in a low volume of PBS (50-100ul) at room temp. Fill a cryomold with OCT (400-450ul). Get all your cells in a syringe (no air bubbles). Slowly inject the cells into OCT while gently stirring the needle. Freeze and later cut using a cryotome. Proceed with IF staining.
Dear Ashwini, Korcan suggested a very good method, but you could also try it with additional modification. Prepare your PBS with 20-30% sucrose (easy way is to prepare sucrose solution and use it to dissolve a PBS tablet - which are usually either to make 100 or 200 ml of PBS) and use 50-100ul of this solution to re-suspend your cells. This is the simplest cryoprotection method which works very well for fixed tissue before embedding in OCT for cryosectioning. For tissue cryoprotection some methods call for 20, some for 30% sucrose. Good luck with your project.
Have you tried any protocols including polycationic coating on your slides, e.g. poly-l-lysine?
Thank you Korcan and Maria...but is it really necessary to have cryosectioning for fluorescence microscopy?...because there will be single cell suspension...
Thanks Charles...I had tried attaching cells with poly-L- lysine...but attachment was very low....can I complete all staining procedure and then attach cells to glass slide?
I think that completing the staining procedure before attaching your cells is preferable, otherwise the coating could catch one of your fluorophores and yield a noisy background.
If you can fix your cells, you will be able to get them to attach to the coating for a longer time.
I hope this helps.
The optimal would be spinning, which you can't do because of lack of equipment and cyosectioning leaves you with dispersed cells. Your main problem is the equipment, so I have tried find a method without cytospin or cryosectioning. Have you asked for help a local pathology/haematology department? - if they don't have cytospin they should have a centrifuge. If they let you use it you could use a modification of the protocol for preparation wax blocks from John Hopkins School of Medicine which I included below. You could follow the entire procedure or modify it. If you search other links embedded in this text (you might copy and search using strings of words via Google) you will learn about other available techniques to deal with cells which may help you with your project.
http://ac.els-cdn.com/B9780124200678000180/1-s2.0-B9780124200678000180-main.pdf?_tid=08cd5406-95bd-11e4-ab76-00000aacb361&acdnat=1420560165_abd87e826146af2ff8c7be1e8b07ce33
Thank you so much MARIA...I will have a look at the procedure mentioned in the paper...
Although it's too late for you, I would like to recommend you to use Smear Gell which is the product that can fix cell suspension on slide easily.
For more detail: http://www.diagnocine.com/download/ipgell-smeargells.pdf
Thank you so much Benjaporn...its never too late..I will have a look on it..Regards
Hello Ashwini, Shi-fix coverslips are an easy option for this purpose. Suspension cells attach quickly on these coverslips ( 30 mins) or can be directly grown on them. The slides work very well for Immunoflurescence.
http://www.shikharbiotech.com/products/shi-fix-coverslips/
Hope that helps you if the issue is still relevant to you.
Regards,
Ravindra
You could try resuspending the cells in mount/Antifade solution after a in- suspension staining.
Hi,
Never 2 late?
I now couping with the same challenge. For now, I stain cells in a plate (96 wells round bottom), then deposit stained cells on a slide (in 10 uL or so). I dry them a bit on the slide at RT. Then I drop mounting medium on them. Then coverslip. Results vary. Some cells look smeared, other cells look perfect. I guess if coverslipping is done more carefully, it might give good results.
What I do not like about this method is that we can not assure the same sample thickness. This shall not be a problem for the confocal microscopy, but for a “normal” one, I guess it might be a problem.
Previously I done tissue sections in OCT or gelatine. Cells/tissues stick very well to the glass slide in those media. I think may be I shall drop the cells in gelatine on slides and then remove the gelatine as I did with sections... I wonder if it would attach cells to the slide in the uniform fashion?
Hi Kateryna,
It's nice idea to put cells on the slide. I am just wondering if I can direcltly re-suspend cells with mounting medium?
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