Ki-67 staining of cell culture

[gussy]

Dear Forum,
 I would like to stain my cell culture with Ki-67,but I dont have experience before to stain from cell culture,should I put my cell on the slide glass? Is it also need fixation? Any suggestion will be very helpfull

Thank you very much

[excalibur]

What are the cells grown in/on?

Yes, you will still need fixation.

[gussy]

The cell is C6 glioma cell line which grows adhere to the basement of culture flask

[excalibur]

You have several options. You just need to decide which one you want to use.

You can scrape the cells you have, spin down to a cell block, fix and process to paraffin.

Or, scrape the cells and use a cytospin centrifuge to make cytology slides. Fix and stain.

Or, grow cells on coverslips. Fix and stain.

Etc. Etc. Etc.

[gussy]

Thank you, I usually subculture mycell by detach them by tripsin EDTA and then centrifuge and I get pellet of my cell and I throw the supernatan,can I use this for making cytology slides? and then what fixation should I use?once more do I also need to perform retrieval antigen.Thanks before

[richard03]

Since you have cell pellet, you may proceed with paraffin embedding as excalibur suggested.

You may want to fix the cell with formalin and then embed the cell pellet with HistoGel which can be purchased from "Richard Allan Scientific"

The following page has some detailed info about the use of HistoGel. Go down the page and look for "Preparation of Cell Culture Block for Paraffin Embedding Using HistoGel"

http://www.ihcworld.com/_protocols/general_ICC/cell_prep.htm

[richard03]

Or refer to Dr. Giorgio Cattoretti's method.

http://www.ihcworld.com/_protocols/histology/cell_block.htm

[excalibur]

Also, if you don't have or can't wait to order Histogel, plain agar works the same.

[gussy]

Oke, thank you very much

[ImmunoNYC]

The easiest thing to do is plate them on chamber slides (you might need to coat) then let attach and then fix in 4 percent PFA for 20 min. Permeabilize in 0.05 percent Tween 20 and proceed with staining protocol.

The paraffin pellet will also work just perhaps more labor intensive.

In any case make sure whatever antibody you have will work in either prep as that should make the biggest decision about which method to follow.