The hydrophobic pen sometimes create a bigger circle than you expect, it's repells the liquid from a bigger area than the green line. Don't make it too narrow around your tissue.

Jason,

I am not sure that I totally agree with your contention based that it is not a fixation issue.   Histological stains and antibodies are totally different creatures.  Obviously, they both stick to things but I think that is where the similarity ends.  I would suggest that you evaluate some different antigen retrival strategies.  We have found in the past that mouse tissue is highly vulnerable to over fixation and this can result in zero labelling even with excellent antibodies.  If antigen retrival does not work than I think it would fair to blame your hydrophobic pen. 

Below are three antigen retrival protocols that have worked well for us in the past and I would suggest running them all at once on the different slices from the same brain:

EDTA protocol

Rinse in PBS for 5 mins

Boil in EDTA 1mM pH 8.0 for 20 mins @100 C

Rinse in PBS for 5 mins

Sodium citrate protocol

Rinse in PBS for 5 mins

Incubate in 10mM (2.94g in 1L h20) sodium citrate solution pH 8.5  @80 C for 20mins

Rinse in PBS for 5 mins

Pepsin protocol

Rinse in PBS for 5 mins

Incubate in in pepsin solution (1mg/mL in 0.2M HCL – prepared by Zidan) @37 C for 10 mins

Rinse in PBS for 5 mins

Best,

Rohan.

The hydrophobic pen sometimes create a bigger circle than you expect, it's repells the liquid from a bigger area than the green line. Don't make it too narrow around your tissue.

I agree with Vivica, the hydrophobic marker spreads and sometimes covers the tissue even though it seems covered with liquid.

Haematoxylin is somehow not as sensitive towards this.

The fact that it's only a few sections per batch that have this issue would point to the pen as the most likely culprit, the tracing probably came too close to the tissue in these. If it was a fixation or retrieval issue, it shouldn't affect only the edge, of only a few sections, especially if all tissues were fixed and processed in the same way at the same time.

Thanks all, I've tried drawing huge ink circles around the sections and also using Tween 20 in PBS rinses/antibody dilutions to reduce surface tension and the problem didn't occur. That was just one batch though, fingers crossed it doesn't happen again.

Hi Jason

Sometimes I have encountered a similar problem with my sections and I had also initially blamed the barrier pen. However, I have also encountered it when I didn't use the hydrophobic barrier pen and I now think that this "edge effect" is more likely to come about when no surfactant is used in the wash buffer. In my opinion, I think that your reported improvement is due to the addition of Tween 20 as I have found similar improvements myself.

With the ABC kits I would guess that the blocking serum would help the antibody spread over the slide, though if this is pipetted on then the same problem can happen with it (i.e. it retracts from the edge of the slide). Therefore, if you want to avoid using Tween or Triton then just ensuring that the serum covers the slide with a pipette tip (or putting more on around the edges) is sufficient (this has worked for me).  

Dan  

Addition of detergent is definitely good and necessary, but be aware that these hydrophobic pens are not always detergent-proof, especially not against Triton X-100 and tend to leak sometimes... I don't know why the manufactures can't change that. I talked to one once, reporting that the circle "opens" sometimes, but when I told him that I use detergent, he said something like "That comes from detergent, that's normal"...

The image shown suggest that first  antibody mixtures used did not fully covered the section during the incubation phase. It is a simple problem to correct. Just make sure that you add enough of the mixture to cover all the section on the slide.