Sure, take a look at these 2 images. In skeletal muscle, sections were not fixed with PFA, and I had nice results, as you can see. But in bone marrow (which was incubated with 4% PFA for 2h), there's no signal at all.

And yes, the antibody didn't work in your case , too.

Why do you fix your bone marrow samples so long (24 h) in PFA?

Hi Matthias,

I'm afraid, but I got these samples already fixed this way, frozen, and stored at -80°C. Basically, I have no other choice than trying to get the best out of those, even if conditions are not optimal. So my hope is that someone already used this antibody on PFA fixed tissues, and found a good antigen retrieval method that can be applied to make this antibody work, or that someone can suggest another anti-CD31 antibody that works on PFA fixed tissues.

In any case, for the future which is the time that you suggest to use for an in toto fixation of a mouse femur (by the way, the samples I have are not simply bone marrow, but the whole femur bone, bone marrow included)? In my experience with muscle, I used a 2h fixation in 4% PFA, but it is not easy to get a good fixation, since the inner part of the muscle remains unfixed.

see: http://lmrt.mit.edu/publications/2009/March_et_al_Hepatology_supplemental.pdf

page 9

What Fluorochrom do yue use with you antibody

Thank you for the link. I saw that the antibody used it's a BD Pharmingen, do you know which is the clone? I'm a little bit confused, anyway: in the methods paragraph it's stated that a mouse anti-CD31 antibody was used, but then the staining in supplemental figure 3C was done with a different one (rat-CD31, the colours in the legend and in the figure are inverted, by the way).

My primary anti-CD31 antibody is unconjugated, while the secondary antibody I used is an anti Hamster-Alexa 549 made in goat.

Hi

I only know DyLight 549 or Alexa 546. But anyway for both you need a green laser (%60 nm). Do you use such a green laser for exitation?

Don't worry about secondary antibody and its conjugate! They both work fine (I mistyped the Alexa, that is 594).

In the meanwhile I tried some HIER protocols, with the following buffers:

- 10mM citrate buffer pH6;

- 10mM Tris, 1mM EDTA pH9;

- 0.5 M Tris pH10;

preheating buffers and then microwaving for 20 mins.

I also tried a PIER method, with proteinase K, following a method found on IHC world (www.ihcworld.com).

None of the above worked... So I guess I should give up, with this antibody...

sorry i have no new idear but its known thats a big problem with samples fixed so long in PFA

Hello stefano,

I have worked with cd-31 antibody staning for cartilage tissue.

and sadly, your samples has been fixed too long in PFA, which may have resulted in total masking of antigens positive to cd-31.

Paraformaldhyde are crosslinkers, and forms intermolecular bridges normal through free amino groups, hence creating a network of linked antigen, and thus overfixing slides may lead to no immunoreactivity/positvity.

If you have not already tried, use Proteinase K ( from Dako) to retrive those linked antigens.

Apply proteinase K for 10 minutes (in the first instance), as one of the major problem of Proteinase K is it tends to detach the samples from the slides, then wash it in PBS, and apply primary antibody.

Hope this helps.

Polly

Hello Polly,

as I mentioned above, I already tested proteinase K (10 ug/mL), but it doesn't work. You're right, incubating longer than 10-15 minutes, sections detach. Also increasing the concentration to 20ug/mL doesn't ameliorate the retrieval (actually, it worsens the morphology).

Thank you in any case for your suggestion.

Hi can anyone suggest an alternative method for antigen retrieval of CD 31 and VE Cad for paraffin blocks. as of now O tried Cit Buffer pH-6, Proteinase K and Tris Buffer pH-10. Please give insightful suggestions??

Harish, I don't know if this can help you, I tried many of the AR methods proposed in the attached paper, including Tris buffer pH10, none of which actually worked in my case... maybe you'll be more lucky!

Hi! I have the same problem because I'm also using bone samples that were fixed, decalcified and then paraffin embedded.. Did you have any luck in finding the right antigen retrieval?

Gloria,

I have not been able to find a solution to fix this issue!

The only way I found to overcome the problem, was to use an anti-laminin antibody, and to incubate sections with the primary antibody at 4°C for several days (4 at least, but a couple more gave me even better results).

hi there

I am having similar issue with 4% PFA fixed 15 min muscle section.

 Stefano Ciciliot can you shear some images for the cd31 with us thanks.

Sure, take a look at these 2 images. In skeletal muscle, sections were not fixed with PFA, and I had nice results, as you can see. But in bone marrow (which was incubated with 4% PFA for 2h), there's no signal at all.

And yes, the antibody didn't work in your case , too.

thanks for the prompt replay.

what did you use for muscle fixation? methanol or acetone ?

it that ok to share your skeletal muscle protocol 

Thanks again 

You don't need to perform any kind of fixation. This is the protocol I used with anti CD 31 (MAB1398Z, Millipore):

1)      Air dry the sections for 15 min;

2)      Incubate sections for 2h at RT with primary antibody (1:200), diluted in PBS + 0.5% BSA;

3)      Rinse 3x 5min with PBS;

4)      Incubate sections for 1h RT with anti-hamster antibody (1:200), diluted in PBS +0.5% BSA and 2% goat serum;

5)      Rinse 3x 5min with PBS;

6)      Mount

Hi Stefano,

I am have similar problems with bones immunofluorescence. Femurs from 8 week old mice were fixed with 4% PFA overnight. Then they were decalcified with 10% EDTA for 3 days, followed by ON incubation in 20% sucrose. Then the tissues were embedded in OCT. I have obtained 8 micron sections. Which laminin antibody did you use for the blood vessels and what concentration? I have tried PECAM and MECA32 and none of them worked. 

L9393 rabbit polyclonal from Sigma, 1:50 dilution (in PBS-BSA1%). As I mentioned, it gives good results, but you need to incubate the sections for several days at 4°C with the primary antibody.

By the way, I tried to use the MECA32 antibody, too. And it didn't work, just like in your case.

Hi Stefano,

Try to  stain for endoglin; CD105 Biolegend 4 μg ml−1, 120402. It works very well on BM. It nicely label vessels.

Hi Stefano,

Thanks for responding. I tried incubating my sections with anti Laminin ab from Abcam for 3 days at 4 degrees. That did not work very well either (too much non specific staining). I will order the clone from Sigma that you mentioned.

Thanks

Kasturi

Thank you for your suggestion, Elmina.

Kasturi, you're welcome. The attached picture shows you which is the kind of staining that I get with Sigma's anti-laminin antibody, in the diaphysis of mouse femur.  You can clearly see many sinusoids, and a couple of arterioles.

Stefano

Hello Stefano,

That looks great. I don't see anything like that with Abcam laminin Ab.

Thanks for your help

Hi Stephano,

I have one more question about your Laminin IF protocol. Did you use it on decalcified bone sections?

Yes, in 10% EDTA for 3 weeks at 4°C

Hi all, finally could you use the CD31 to stain paraffined slices of tissue? Thanks a lot

Hi everyone,

I used the endoglin (CD105) antibody (suggested by Elmina) on decalcified bone cryosections and and I definitely see sinusoids, but no arterioles as in Stefano's image. I will try the Sigma laminin which he suggested. Another problem I am having, is autofluorescence in the 488 and 594 channels. So can we only immunostain in the Cy5 channel to get good images of bone marrow?

Jordi, I gave up with CD31.

Kasturi, you're right, usually autofluorescence in those two channels is higher than in Cy5 channel. I don't know how much familiar you are with other tissues, but I can tell you that skeletal muscle is even more auto-fluorescent than bone/bone marrow.

In any case, it is always a matter of signal/background ratio. If your signal is good, and you have appropriate controls, you can usually get good results, anyway.

Hi Stefano,

This is my first experiment with tissues. So I am not familiar with these factors. Like you suggested, I am trying to increase the signal to noise with TSA amplification kit.