New to this way of doing IHC. Growing cells on cover slips and looking for phagocytosis on colored particles. Problems encountered 1) cells seems to grow on both sides of the cover slip. 2) too dense packed cells to count. Any ideas to improve?
Hi Mayur,
I don't have a lot of experience with IHC myself, but typically, using 200 ul suspension containing 20,000 cells (adherent cells) has worked for us in the past. We ensure that the cell suspension is dispensed as a drop, and leave it undisturbed for three to four hours before adding any more media. Thus, any remaining unattached cells usually don't detach below the coverslip or anywhere else in the dish. Does that help?
Hi Mayur,
Otherwise, if it's feasible, preparing cytospots using cytospin is also a good option worth trying. Good luck!
http://www.ihcworld.com/_protocols/histology/cytospin.htm
Hi Mayur,
The simplest way of preventing cells from growing under your coverslip is to have the coverslip dry and in the well before you add the cells. Sterilise your coverslip by your method of choice (eg UV sterilisation, autoclaving, EtOH then sterile water wash and dry) before placement into dry wells. You may then want to add a drop of FCS to the top coverslip, as the cells often like this. Then seed your cells at an appropriate density ie if you find that you have too many cells to count, then don't seed as many (assuming that the cells are not contact sensitive - and phagocytic cells usually are not). I've prepared SMC, EC, macrophages, fibroblasts and cell lines in this way for later IHC.
Good luck!
Anita
Hi Mayur,
So what if your cells do grow under your coverslip? When you've finished your experiment and mounted the slip on a glass slide, simply polish the top surface (which WAS the underside) to remove the unwanted cells. As for cell densities, just seed fewer cells per well, as Anita said.
Here's another tip: if you're doing IHC use a plastic coverslip slightly larger than your glass one. These are easily made from a thick polythene bag. you can use as little as 15ul per coverslip which will save on antibodies. They are easily removed by "floating" them off with a little wash-buffer.
All the best,
Simon
Thanks for all these answers! Were great tips. I found out the in addition to a small # of cells, the salts in the media were totally covering the other side of the cover slip, simply wiping it solved it all. I had the gut feeling to perhaps plate less # of cells which some of you already pointed. Thanks you all!
Why don't you use chamber slides? You can do the experiment on the slide, and stain the whole experiment for IHC at the same time. The slides are sterile so there's no problem with sterilising your cover slips. It's also much easier to process the slide rather than a coverslip.
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