This is a rather simple but tedious task. The steps are:
1. Fix cells using 100% ethanol. (This is to prevent the cells from washing off, during the wash step)
2. Incubate for ~20 minutes in the freezer.
3. Remove ethanol and wash thrice using PBS.
4. Add 4% blocking solution and incubate for 1hour on rotator at room temperature. (Blocking solution used here is just BSA, bovine serum albumin)
5. Wash thrice using PBS to wash off any unbound BSA.
6. After 1 hour, add primary antibody (Dilution ratio--> Ab: BSA = 1:100) and incubate on rotator for 1hour at room temperature/incubate overnight in freezer.
7. Wash thrice with PBS to wash off any unbound primary Ab.
8. Add secondary Ab (Dilution ratio--> Ab:PBS = 1:100) and incubate for 1 hour on rotator at room temperature.
9. Wash thrice with PBS.
10. Add DAPI and incubate for 5mins on rotator at room temperature.
11. Wash thrice and proceed to view under fluorescent microscope.
Note: DAPI stains for DNA(Nucleus). It will appear blue under microscope.
The antibodies that i use are:
1. Rabbit anti-ZO-1 (Primary Ab)
2. Alexa Fluor 594 goat anti-rabbit IgG (Secondary Ab)
Rabbit anti-ZO-1 will bind to the ZO-1 protein. This ZO-1 protein can be found only when the cells are in contact with each other.
Alexa Fluor 594 goat anti-rabbit IgG is derived from goat and binds specifically to rabbit Ab. Therefore, when the above two Ab are used together, Alexa Fluor 594 goat anti-rabbit IgG will binds to rabbit anti-ZO-1. When view under microscope, the protein will appear red.
Below are some of the photos i taken using the microscope:
Note: Permission were taken from my mentor before the posting of these images, except for the last image. The last image was taken from website: http://www.biotechniques.com/BiotechniquesJournal/2007/August/Murine-nasal-septa-for-respiratory-epithelial-air-liquid-interface-cultures/biotechniques-43042.html?pageNum=4
The yellow box shows the ZO-1 protein that I'm talking about. The reddish thing is the cytoplasm, which is overstained. But for the image below, the distinct red lines are the ZO-1 proteins. The green stain is type IV beta-tubulin stain, according to the website.
Reference:
BioTechniques, 2009. Retrieved on 27 August 2009 from website: http://www.biotechniques.com/BiotechniquesJournal/2007/August/Murine-nasal-septa-for-respiratory-epithelial-air-liquid-interface-cultures/biotechniques-43042.html?pageNum=4
Alvin
Hey Alvin,
ReplyDeleteCan I know why is it that the initial incubation is in the freezer?
Thanks!
Siew Ming
TG 01 Group 2
0702862D
Hello Alvin,
ReplyDeleteWhat is the purpose of detecting the ZO-1 protein of cells in contact with each other?
Jeremy
TG01 Group 1
0702919B
Alvin,
ReplyDelete(1)Why are your primary and secondary antibodies dilution different from one another ie. Primary in BSA, Secondary in PBS.
(2)What is this ZO-1 protein?
(3)From what I observed from your pictures, some of the individual cells have red fluorescent staining around them. So does that mean that ZO-1 protein is present on individual cells ie not in contact with other cells?
Li Yinliang Alex
0704894E TG02
Group 8
26/08/09
Hi alvin~
ReplyDeleteis any detector uses in this case or will DAPI flourescent once it's bound to the secondary Ab?
Also, why can't you just add a DAPI labelled secondary Ab like what we've learnt?
thanks alot!
eriko
0700477C
Siew Ming:
ReplyDeleteI asked my mentor why is it in the freezer. He said: "I'm not sure too, its found in the protocol i used for the first time."
However, we guess that it may be to inactivate the cells to completely stop its growth.
Jeremy:
By detecting if ZO-1 protein is present, we will know if the cells are in contact with each other. It is important for my project as i need the cells to be in close contact with one another. This eliminates any possible gaps formation in between the cells that may cause some leakage.
Alex:
As the previous step of incubating with BSA may not be sufficient to block off all background effect, the primary Ab is diluted in BSA to ensure that that doesnt happen. Secondary Ab can be diluted using BSA too, no problem. But BSA is cost more. Therefore, PBS is used.
As i said in my post, this ZO-1 protein can only be found in cells that are in contact in each other. So it is something like a cell contact protein.
As i was rushing to post the blog the other day(for some reason), i didnt post a description on the images. But i have edited them. The ZO-1 protein is the one in the yellow box i drawn. The image may be rather small to see. Therefore, i went online to search for a clearer image that show distinct ZO-1 protein. Take a look in the post.
Eriko:
DAPI doesnt bind to the secondarty Ab. It binds to the DNA. Therefore, when it is view under a fluorescent microscope, it will fluoresce.
Did we learn something on DAPI labelled secondary Ab? Which module was that? Cant remember. Anyway, i asked my mentor on that. He said there isnt any secondary Ab tagged with DAPI.
Alvin
hi alvin!
ReplyDeleteyour method, is very similar to mine! is it possible to use Hoechst staining instead of DAPI too?
LIM JIA HUI JOEY
tgo1 0703605F