OX 42/CD11b

[cincanada]

I am trying to do immuno staining on rat brain.  I have tried a FITC labelled CD11b (no luck) and the standard biotinylated ABC DAB with no luck.

Any suggestions?

Block with 3% BSA
1AB in 1%BSA
wash in PBS
2AB in 1%BSA
wash in PBS
ABC
wash in PBS
wash in Acetate buffer
DAB
wash in Acetate buffer
wash in PBS

Any idea of what I should see?  The images I have seen are not really that clear.

[excalibur]

Found this on Abcam's site about your antibody:

IHC-P: 1/25 - 1/200. Use on periodate lysine paraformaldehyde fixed paraffin embedded tissues (the antibody does not work on paraformaldehyde-only fixed), perform enzymatic antigen retrieval before commencing with IHC staining protocol.

Check you fixative and perform antigen retrieval.

[cincanada]

Unfortunately the tissue that has been collected was perfused with 4% PFA.  How can I possibly get the OX42 to work?  I am on a time limit as this is suppose to be my summer students project and I am so busy I do not have time to trouble shoot with him.

Suggestions?  I do not know how to do antigen retrieval.

C :oops:

[excalibur]

Well, all may not be lost. It says to do enzymatic AR, which is using something like protease or trypsin. Do you have the data sheet that came with the antibody? If not, the company should have it on its website as to recommendations for your specific antibody.

Here is a link to this websites AR techniques:
http://www.ihcworld.com/epitope%20retrieval.htm

and here:
http://www.ihcworld.com/_technical_tips/antigen_retrieval_tips.htm

[Erebus]

Dilute your primary ab in BSA + TritonX-100/saponin for enhanced tissue penetration. If i remember correctly no ar was really needed. Did it a couple of years ago with alexa 488.

I know a couple of oxperts if you need more help.  

OX-42 for a summer project. That is mean.

[cincanada]

Hmmmmmm, was not my idea for the summer student (my investigator picked the project) I am just suppose to help him.

Thanks, I will give these a try and hopefully have the bugs out before he gets back next week and I leave on vacation.

C

[cincanada]

What was the concentration of the BSA + Triton X/ Saponin?

C

[Erebus]

I would go for 1%BSA and 0,3%Triton X (not sure about saponin). I think i did immunofluorescence with alexa488.

Good luck!

[cincanada]

Thanks.  I have never used Saponin before so I have no idea what to use....suggestions?


C

[cincanada]

Oh, and do I have to heat it?

[Erebus]

Skip the saponin. You donīt have to use them both. I am not sure about the heating. I think people around here have been using a microwave for that. I didnīt.

[cincanada]

Hmmmm, it did not work.  Either protocol.  I tried both that have been suggested.

Any other suggestions?

[excalibur]

Fixation. Fixation. Fixation.

[F.Bernal]

I don't know if you still are on time, but I would plainly change of antibody suplier. I have used CD11b from Serotec with very good results in cryosectioned tissue postfixated, so I guess it would also work with perfused tissue. If you need fluorescence you can always use an apropriate secondary Ab...