I am imaging some optic nerve head sections using non linear microscopy (SHG/TPEF simultanously). I am going to merge the two channels (SGH green and TPEF red) in order to see the co-localisation of collagen and elastin. I am getting some autofluorescence from TPEF coming from the whole sample so it makes difficult to distinghuish elastin from everything else.
What can I do to avoi/remove/reduce auto fluorescence?
Thank you
Thank you
This sounds helpful. However I am not sure what you meant with "You will need to fix, permeablise and block sections all experiments simultaneously".
Can I ask you to explain a bit?
Thank you very much
Regards
Laura
Hi,
let me explain how I prepared the samples. As I am in the first year of my PhD I had some optic nerves kept in PFA that have been collected few years ago. Anyway, I removed the sample from the PFA and I cut 250 microns thick longitudinal sections with a sledge microtome and I put them in a cling film, stored at -80 until imaging.
Then I thaw the samples and once they have reached room temperature I put them in a petri dish with some PBS: glycerol 1:1. I used an immersion lens as we are trying to develop a new method for non linear microscopy using immersion lens. But the auto fluorescence has been seen with a normal lens too (maybe it was less intense), using a positive control for elastin.
Is this what you were are asking for?
Please let me know.
Kind regards
Laura
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