cells stained with NF-KB and DAPI - (Sep/08/2006 )

Hi,

I would like to ask for help from your guys again. I want to check the translocation of NF-KB in cells. I know I can do NF-KB immunofluorescent staining and also stain the cells with DAPI to localize the nuclear. However, I never have any experience about cell staining. Could somebody can give me some reference or some suggestion?

Thanks for your reply!


Blue-snow

---

Hi Blue-snow,

You may check "Flow cytometry: a practical approach", Edited by Michael Ormerod. Oxford University Press. Otherwise, I you may check two papers I found in the Medline (unfortunately I have no subscription online to send you the .pdf files). Here they go, anyway:

1: Lee SY, Ha TY, Son DJ, Kim SR, Hong JT. Effect of sesaminol glucosides on beta-amyloid-induced PC12 cell death through antioxidant mechanisms. Neurosci Res. 2005 Aug;52(4):330-41. PMID: 15885833 [PubMed - indexed for MEDLINE]

2: Nadjar A, Tridon V, May MJ, Ghosh S, Dantzer R, Amedee T, Parnet P. NFkappaB activates in vivo the synthesis of inducible Cox-2 in the brain. J Cereb Blood Flow Metab. 2005 Aug;25(8):1047-59.
PMID: 15758944 [PubMed - indexed for MEDLINE]

Hope this helps. Regards,
Gerardo

---

I have some experience in immunocytochemistry (if you want to stain your cells with DAPI I assume it's for icc). If I had to do your staining, I certainly would proceed like that:

1. discard the culture medium (after eventual cells treatment)
2. wash 3x 5min with HBSS (just lay HBSS on your cells, and change it after 5min.. do it 3 times)
3. fixation with paraformaldehyde 4% (15 or 20min)
4. wash 3x 5min with PBS
5. permeabilization of cell membranes (necessary for intracellular antigens staining) with Triton 1% (i'm not sure of the incubation time but i'll ckeck it if you're interested)
6. wash 3x 5min with PBS
7. blocking (to avoid aspecific binding of your secundary antibody) with PBS BSA 5% (or serum from the animal in which the secundary antibody was produced) and Triton 1% for 30 min at RT
8. primary antibody, diluted in PBS BSA 1% Triton 1%. incubation time and temperature will dependent on the affinity of your antibody for the antigen. usually we try first 1 hour at RT (to enhance antibody/antigen binding, you can work à 37°C and/or let the antibody for 2 hours)
9. wash 3x 5 min with PBS (washes are really boring, but necessary ;-) )
10. secundary antibody, diluted in PBS BSA 1% Triton 1%, 30 min or 1 hour at RT (protect from light to preserve the fluorescence!). at this step you can also add DAPI, if it's not include in the mounting medium.
11. wash 3x 5min with PBS (courage! last washes!) (protect from the light!)
12. mouting (we use mounting medium with DAPI).

after that, always protect your slides or dishes from the light!
I go quickly to the fluorescence microscope ;-)

I hope it will help you...

---

Just an important specification: if you work on slides, all your incubations (blocking and antibodies) have to be done in an humidified chamber to avoid drying of your prep! I will explain you how to make an humidified chamber easily if you're interested..

---

beside triton x-100 we add saponin to permealize cells; 5 min will do; there are humidified chambers to buy but incubate in a light-protected box with high humidity by adding water-wet sponge or cellulose paper

---

Hi Gerardo,

I can only one paper(NFkappaB activates in vivo the synthesis of inducible Cox-2 in the brain). In its material and methods parts, they gave some information about the immunocytochemistry. I think it is too simple for me as I never have any experience about cell staining. I would like a detailed protocol from someone have worked . Thank you for your help!

Blue-snow

---

Hi behappy736,

Thank you very much for sending me a nice protocol. But I still have some questions:

1. My cells are adherent cells, should I grow my cells in cover slides(Poly-L-Lysine coated) or just harvest it as pellet and finish the staining process?
2. How can I mount the single cells (I only know how to mount drosophila embryos)?

---

I am interested in making a humidified chamber, could you please give me more details? Thanks!

---

Hi kosmodrom,

Thank you for your suggestion!

---

1. For staining of adherent cells we use 3 kinds of cell support:
- normal multiwell dishes (coated or not, depending on the cellular type)
- or cover slides in multiwell dishes
- or Lab-Tek (from Nunc), which is multiwell mounted on a slide. To me, this is the easiest. You do the staining like in a normal multiwell dish (here, humidified chamber is not needed) and, just before mounting, you discard the "walls" with an supplied device then you get just the stained-cells an microscope slide (without any perilous handling!).

The problems with normal multiwell dishes are (1) that mounting might be perilous, (2) that you need an inverted microscope, (3) that storage is not easy. But for a preliminar test it might be right.

I'm not familiar with the use of cover slides, I know handling is sometimes no easy. But if you know how to use it, it's ok too.

We don't use Poly-L-Lysine, because our cells don't need it to adhere on our supports. I don't know what about your cells...

Don't harvest the cells!

2. Mounting single adherent cells is just like mounting an histological section (with an aqueous mounting medium! not xylol-based medium).
1 or 2 drops of mounting medium on the sample, then laying of the cover slide (while avoiding bubbles formation). And, if needed, fix the cover slide on the microscope slide with nail varnish (if you're working with Lab-Tek).

p.s. in protocol: it's not Triton 1%, but Triton 0,3% (everywhere), and the incubation time for permeabilization is 20 min

Bye!

---