I am working on 30um sections of adult drosophila brains. I dual stain the sections first with Alexa-fluor 594 anti-HRP stain and then with DAPI to stain the neurons and the nuclei respectively.
Right now, after (cryo)sectioning, I capture the sections on Superfrost GoldPlus slides, add few microliters of the staining solution onto the slide and cover it with parafilm for uniform spreading of the stain. I do the same procedure for washing with PBS, however, the sections get untethered from the slides
1. When peeling the parafilm away, the section gets stuck to the parafilm (or)
2. The section begins to float while washing with PBS or adding the staining solution.
How do I prevent this from happening?
Hi,
i think use of parafilm can be cause of the problem. you should not use parafilim . also you can use of charged slide to increase sample adhesion on slide.
good luck,
I do have access to a confocal. However, the objectives are not that powerful; they can penetrate only to a maximum of 60-70 microns.
Thinner slices might aid in adheshion, but will make the tracing out of laminar neurons (which I am interested in) much more difficult.
I think choosing not to leave parafilm on the sections is indeed a good idea and I will try it. One problem that might arise though is that - the tissues might dry out. I will use a moisture chamber to counter this problem.
I don't have PLL-coated slides in my lab; will look into it.
Hi Rathnavel,
Did you verify if your slides are good? We are using pre-cleaned Superfrost Plus slides and the quality can differ from lot to lot. Also instead of Parafilm you might want to consider using a PAP - pen to outline your sections, to keep the solution on the sections.
Best Simone
Hello Simone.
Thanks for your input. The slides seem to be good. Is there, by any chance a grading scale for the quality of slides? The idea of PAP-pen seems to be promising and I will utilise it. Thank you.
Hi Rathnavel,
As far as I am aware, there is not really a grading scale for slides. The way I test my slides is by testing if and how a droplet of water spreads on the slides. If the drop of water spreads all over the slide and does not drip off easily, when you tilt the slide, the charge is not good, hence your sections are probably not going to stick. If the droplet does not spread and drips off easily when the slide is tilted, then the charge is good.
You also might want to have a look at this website.
http://www.ihcworld.com/_technical_tips/section_falloff_tips.htm
Best Simone
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