Dear colleagues,
In my immunofluorescence stainigs I use liquid blocker pen, before solution with 0.3% Triton X-100. This helps to avoid dripping of solutions with primary antibodies and Triton (24h incubation in water bath) from the sections . But I suspect this pen may enhances the background.
Does anyone know how to fix this problem?
Thanks for attention
Anastasia
Hi Anastasia,
There should not be any problem with this pen. I use a pen from Vector Lab for variety of immuno-stainings but never had a problem. Only problem was if I did not dry it enough after drawing, a liquid was leaking.
Hi Anastasia,
Triton X-100 should not affect most hydrophobic blocker pens. Are you allowing it enough time to set? Personally I do this between a PBS wash (after antigen retrieval, but before any incubation that involves leaving a slide out of a PBS/H2O bath), setting it out with enough PBS covering the section, drying round the edges with blue-roll, marking, and then allowing it to set. I would consider using the Triton X-100 earlier in the protocol as you should only need it once to permeabilise.
The most likely suspect would be over-permeabilisation with the extended incubation with the Triton X-100. Depending on section thickness and concentration a much shorter incubation (in the range of 10 minutes) should be sufficient, then if still necessary switch to a gentler detergent, with something like 0.1% Tween-20 for the remaining buffers. But this is difficult to know without seeing your protocol/section type.
If you're using thinner sections (e.g. 3-5um) then you can do without permeabilisation at all.
Hi,
As Andrey Kostin mentioned, I think you should let it dry a bit before adding your antibodies. The other thing is when you draw a circle around your sections, be careful not to push the pen down so much as it might give you background later on.
Good luck
Thanks, Edward Fielder
I make 20 um slices of the mouse brain and use Triton for stainig on a second marker. Before applying, I also wipe the glass around the edges and wait for the marker to dry. I did not associate the appearance of the background with excessive permeability, however, I also label on intracellular markers. Thanks for this idea, I will try to change the protocol next time. What is this blue roller?
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