In unpublished work on the choroid plexus in mice we used this protocol to stain 4%PFA fixed brains:

Tight junction staining of choroid plexus of the lateral ventricles:

• Cryosection, 10 um, onto glass slides
• Wash with PBS with 0,3% Triton-X for 15 min
• Incubate with Rabbit polyclonal Anti-ZO1 antibody (1:200) 4c overnight in PBST
• Wash with PBS  10 min x 3
• Incubate with Donkey anti-rabbit Alexa564 (Jackson, 1:500) and DAPI (1:1000) in PBST with 5% Donkey serum overnight
• Wash with PBS  10 min x 3
• Mount in Prolong Gold

This gave us good images but did require

As well in unpublished work, one of my colleague managed to make her anti-ZO1 antibody (Rabbit polyclonal 1:100, Invitrogen) work after some optimizations. She is also working on brain slices and realized that finer slices were indeed better (20 µm) but crucial is also the fixation itself: she flash froze the fresh brains after PBS perfusion (no PFA perfusion) and then performed cryoslices. On this fresh brain slices, she performed a post-fixation with acetone. Trials with PFA fixation were completely unsuccessful. As you can see on the picture enclosed, the staining is, in these conditions, really looking nice. Hope this will help !

Hi Kristian,

Thank you for responding so thoroughly. I will be staining in cortex and hippocampus using an antibody raised in rabbit from Invitrogen. I found a paper from 2005 characterizing how well various antibodies stain for tight junction proteins in mouse brain. The one I am currently using was found to work quite well so I am not too worried about that. As for the tissue, I will have to check on that. That's a good point though. Thank you! I do permeabilize before staining and also have a protocol from a previous lab mate that includes a pepsin antigen retrieval step. I have yet to try that though. Imaging will be done with a Nikon A1 laser scanning confocal microscope which does allow the entire depth to imaged. The thickness just may not be optimal for staining. Along with ZO-1 I will stain for claudin-5 and CD31. I have gotten both of these to work though so no problems there. Thanks again for taking the time to respond.

Hi Florie,

Thank you for responding. It's good to have someone confirm a protocol similar to what I am reading in the literature.

Hej William, do you have a link to the paper you mention?

Hej. Florie, I'm envious of your colleague's stain. I'll try the PBS and post-fixation with actone next time. 

Well, for all their immunocytochemistry problems people mostly blame their fixation or antigen retrieval techniques.

However, in my long experience (about 30 years) I have noticed that the primary antibody is the most important factor. You simply have to have a good primary for your immuno to work. My suggestion is, first try to find a good primary known to be working with PFA fixed paraffin embedded sections. I think most your problems would then disappear. Finding one good antibody is like a gold mine, once you get it, most likely you can make a lot of papers.

Regards

Hi everyone,

I want to say thank you so much for your time and responses. Sorry for such a long delay in my response. I have established a fantastic and highly reproducible protocol. Feel free to message me if you should ever need to stain for tight junction proteins or need a protocol for snap freezing.

Hi, William Mills III

Do you mind sending the protocol for tight junction proteins to me? By now, I am trying some IHC staining with rat brain slices for tight junction in blood-brain barrier, but have difficulties of gaining signals.

Thank you so much!

Hi William Mills III

Do you mind sharing the protocol?

Also, I have been having difficulty staining monolayers of bEnd.3 cells for ZO-1. I tried both mouse mono and poly rabbit for primary antibodies but the staining is dim and not localized to the junctions.

Thanks a lot.

Hi William, Can you send me the protocol for ZO-1 staining in mouse brain sections? Also, can you tell me the company and catalog for this antibody?

Hello, I reduced the fixation agent from 4%PFA to 1% PFA and now the staining works!

Over and over again, I have seen that the main problem for the failure of immuno is the primary antibody. Get a good primary that works with Formalin fixed paraffin sections that works without antigen retrieval and all your problems will disappear!

4% PFA also causes a lot of background. In my view Bouin's fixative is the best. Remember to wash thoroughly to remove the yellow picric acid.

Frozen section is OK, but it causes huge distortion of the architecture of the tissue. Besides, the paraffin sections can be kept for many years.

So, the bottom line is a good primary antibody is the key to get a good immunostaining in paraffin sections.

Regards

Dear Nurul Kabir

so how can we remove background noise in immuno cytochemistr, for which we used 4% PFA?

There are some methods of removing the background but none of them works very well. This is why I always avoided using 4% PFA. I used either the neutral buffered formalin or the Bouin's. Bouin's seems to be the best.

Hi William! Would you mind sending me the protocol you optimized for brain staining of ZO1, please??? Thanks!!!!

Hi William. Would you be ok to sending me the protocol you optimized for ZO1 staining, please ?

Many thanks

Hi William, could you please send me your protocol. I'm having trouble staining ZO-1 in airways of mice. Kind regards, Richard

Hi William, could you please send me your protocol.

Thanks!

William Mills III do you mind sharing your protocol ZO-1 and claudin-5 (if any)

William Mills III Hello William, May you please send me the protocol for snap freezing and ZO-1 staining? I would really appreciate it.

Thanks.

Hi William Mills III ,

Glad to see you was able to fix your staining. I`m currently having the same issues. Do you mind sharing your protocol for staining tight junction proteins and for snap freezing? thank you so much in advance!.

Hi William Mills III , I read that you would be happy to share your protocol for staining TJ.

Would you mind sending it?

Thank you very much

Hi William Mills III , I am having trouble with ZO-1 and CD31 co-staining. Would you be able to send me your protocol?

Thank you!

Hi William Mills III , could you please send me your protocol, I'm having troubles with TJ staining.

Thanks a lot!!