Hi all,
I'm doing immunocytochemistry to detect and visualize my protein of interest in SH-Sy5y cells, however I faced two problems:
First, growing of SH-Sy5y cells on glass coverslips, which many of them washing off during the process. I coated the coverslips with Poly-D-Lysin for 2 hrs before loading cells on them. But I saw many of cells are lifting during ICC. How to prevent it?
Can I use Fibronectin or Matrigel instead of Poly-D-Lysin? Which one is better for my application?
Second, after finishing the process, when I'm looked at slides under fluorescent microscope, the image intensity is very very low, I could not see the proteins at all. Only can visualized cells with DAPI.
I'm using Triton X-100 (0.5%) for cell membrane permeability (incubate for 1hr at R.T), incubate with primary antibody (1:250) overnight at 4C, incubate with secondary antibody (1:500) 2 hr at R.T. My secondary antibody in conjugated with Alexa flour 488.
I appreciate your help in advance
Hi Elham Amini. E.A ,
I have never worked with that specific cell line but maybe I can still give some advice.
Regarding the coating, I would recommend to use the same coating you use for normal culturing. In my group we also use Matrigel on cover slips. Sometimes glass needs a higher coating concentration than plastic ware, so this could be something to try if you still experience detachment.
Are you sure this protein is expressed in the cell line and that the antibody works? So do you have any kind of positive control?
Then of course you could play around with the dilution of your antibody. For the secondary antibody, 1:500 is fine in my opinion.
Furthermore, you might need a higher magnification to see small proteins. So make sure your microscope is capable of capturing it.
I hope this helps.
Good luck,
Selene
Powerpoint has good suggestions for that purpose or try Imgage J software, it is free.
I know many researchers like immunofluorescence and I have used it in the past, but I much prefer immunohistochemistry for many reasons. When doing cell cultures I alsoI prefer using multi-chamber slides instead of coverslips. With multi-chamber culture slides you can also do immunohistochemistry and get permanent brown color that does not fade then capture images with bright field microscopy and digital camera.
Hi,
to help ICC try this protocol:
Dear Selene Lickfett,
Thank you for your reply. Yes, I'm sure this protein is expressed in the cell line. And I will do your suggestions as well.
Dear Hazem Almhanna
Thanks for your reply. Actually, my issue is visualize the protein under microscope. I need to improve its intensity
I got you, so you need to choose positive control for antibody, or then checck your retrieval solution buffer, OR ABC kit, or exposure time for TEMED, also time for counter stains, I am sure you will find the reason if you are sure about your antibodies.
Dear Mamoun Younes
Thank you for your reply. Actually, your suggestion about multi-chamber slides is good, however now I can not purchase it because of price.
So now, there is no way for me to improve cell attachment to the coated coverslips, then improve to visualize them under microscope.
Dear Mateo Glumac,
I have a question that, may I use serum such as Donkey serum or goat serum as a blocking buffer and also as solution for primary and secondary antibodies? Or BSA in PBS alone is better?
Thank you
Elham Amini. E.A
I prefer BSA but you could use donkey or goat serum as well. In that case, I would choose one that differs from the animal in the primary antibody that was raised.
I hope the protocol helps you with your studies.
Best wishes,
Mateo
Dear Hazem Almhanna
Thanks again for your reply. Positive control you mean try another antibody, right?
Dear Mateo Glumac
Thank you so much for your reply and method. I will try this protocol and inform you the result.
Hi Elham Amini. E.A
Although it is a late response, hope would be helpful. I am working with this type of cells. Place your coverslips inside the plate-well (be careful to choose a suitable coverslip size because cells may collect around its boarder), then coat the coverslips with fibronectin. After one hour wash with PBS and seed your cells. Perform the IF protocol, then on a slide put one drop of the antifade for each coverslip, pick the coverslips and place them face down. Now you are ready to take images, if this does not work, the problem maybe in the antibodies ..etc.
Dear Mohamed Khashan
Thank you for your reply. I used the same process except using Poly-D-Lysin instead of fibronectin.
But another problem is visualization of cells under fluorescent microscope.
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