Thread: Looking for the right antibody

Hi all,
My problem is the following: We are making research on an organ made of collagen Type I which also contain cells, and we are trying to find the right antibody to stain it, because it quite transparent and also absolutely unknown by most scientists. The diametere is around 100-200 um and it has a tubular network struktur. Has somebody a goo idea how to find / make the right antibody to stain it ?

THANKS !!

Fred

1. what kind of sections do you have? paraffin or frozen
2. what kind of staining do you want? Fluorescence? Dab?
3. what is the species?

1. what kind of sections do you have? paraffin or frozen
both, we dont care....
2. what kind of staining do you want? Fluorescence? Dab?
Any thing is ok, just as easy as possible
3. what is the species?
Rats, mouse and rabbits we use all of them lets take the easiest one...

One way is to go through publications and find decent collagen type I antibodies and order those.

What I personally like is the service that Labvision offers. You can order any antibody from them in the limited amount of 0.1 ml for 70 euro (depends a bit where you live) per sample. Therefore it is not really a big loss if it doesn't work. I often order multiple antibodies against the same target if they have them and test them all.

But I see now in their 2007 catalog that they only have one collagen type I antibody and it is used for western blot only.

So you will have to look around.

Often the best thing to do is just to google it.
http://www.google.fi/search?q=collagen+type+I+antibody
and then to check out if any of these antibodies have been used by someone else in publications or just take a gamble with one that sounds good.

That said, you say the structure is transparent. But what does that mean? Transparent in a haemotoxylin eosin staining? trichrome? Any other histological staining?

What's easiest DAB or fluorencence? It all depends on the expertise you have, the stuff you have in your lab, and what kind of picture you want to publish.

Showing a structure is better in my humble opinion with plain light microscopy, so I wouldn't touch fluorescence unless absolutely necessary. If you want to do double stainings at one point you might want to do fluorescence though.

Optimizing for DAB for instance doesn't mean you will it optimized for fluorescence so if you are planning to go the double staining route you might as well go immediately for fluorescence.

Immunohistochemistry is still like magic. Sometimes an antibody works, sometimes not. Sometimes you have to optimized it till your fingers bleed. sometimes it works under any condition.

I love it.

and hate it.

I think you didnt get my question, maybe I should have explained more in detail...
I cant take a collagen staining because many other things are also made of collage in a mammal. Well these tubes contain cells and have also membranes, there must be a specific receptor on this structure, the question is more:
how do I find the structrure of this recpetore to have a specific antibody...

But thanks that you explained me how to buy a antibody :wink:

How do you find a specific receptor in a tissue you do not know anything about?

Hard work or collaboration.

Trial and error. I would just start doing in situs with whatever probes we have and hope I would get lucky. But I wouldn't bother doing this to be honest.

If you are working with standard model systems you can try a microarray. See if something sticks out. But then you need to compare your structure with something. That might be a problem. And apparently there aren't many cells in your structure, so you might also have problems with getting enough material for the micro array.

Otherwise contact some biochemists at your university or research institute. See if they are interested in a collaboration. I'm collaborating with one. She isolated a novel protein. Been working her ass off for 2 years to get it solated and generate a specific antibody for it. Still don't know if the antibody works. Probably not. I have to save her PhD by fixing her up with a small paper so she has enough publications. I wouldn't get my hopes up for a nice clean solution.

mainly because your question is biased. the structure is special and hence the cells must be special. And they must have special receptors.

None of these associations are necessarily true.

Ok, thanks for the ideas...
If someone knows something else I would be glad to hear about it...

Cya

way, way too complicated for an answer here. Anything given would be dramatically watered down. If you really want to know, go to:

Code:

www.pubmed.com

click 'books' on the far right, read chapter 9 of Immunobiology by Janeway and Travers.
_________________
Health