Hi friends,
Last week I carried out an experiment to detect the effect of an agent of interest on KCL-induced alpha synuclein aggregations in SH-SY5Y. We used three wells in a 24-well plate as follow:
1st well: KCL-treated and transfected with the agent.
2nd well: KCL-treated.
3.rd: neither KCL nor transfection was applied.
After 48 hours we detected alpha synuclein aggregations by IF and Thioflavin S. The results of IF were pretty beautiful, but the ones of thioflavin were un-readable and to some extent do not match those of IF.
The protocol of thioflavin which I used as follow:
I fixed cells with PFA 4% for 10 min. Then I treated cells with thioflavin 0.05% (2.5 mg in 5 ml) for 20 min. After that, I washed twice with ethanol 80% and one time with PBS for 5 min each.
In the attachment I added 20x photos of my three wells.
The abbreviations of the photos are: TRA = transfected, CTRL = neither KCL nor transfection, KCL = only KCL without transfection.
Any comment or suggestion is welcome.
Thank you in advance.
Hi Mohamed Khashan,
All your images have fluorescence. What do you mean by unreadable? Are you supposed to have 2-channel images? What excitation wavelengths did you use? Filters?
Hi Johanna Marie Dela Cruz
Thank you for your response. Unreadable means uninterpretable. The Control sample should not have fluorescent like the other ones, so I doubt that my protocol does not work well or there is a fault in other step in my experiment.
Regarding your other questions, I used one-channel image and the wave length was 488.
If you kept all your acquisition parameters the same for all 3 images, this could be autofluorescence. You would have to measure the integrated density in each of your images and compare them. Take background into account as well. Also, if your original images were stored in jpg format, the results will not be reliable because data is compressed.
Johanna Marie Dela Cruz thank you very much for these precious information. Please can you explain what do you mean by (If you kept all your acquisition parameters the same for all 3 images)? Should I change the parameter for each photo?
My original images are CZI but I have converted them to JPG.
Mohamed Khashan , if you acquired an image with a certian laser power, gain, frame size, zoom, etc., all your other images from your control or experimental samples should be acquired with the same settings.
Analysis should also be done with the .czi files.
Johanna Marie Dela Cruz thank you very much. I appreciate your comments.
Johanna Marie Dela Cruz Hi and sorry to be off topic, I would like to ask you if you can guide me to a way in ImageJ that help in counting the number of aggregations?
Mohamed Khashan , you first need to segment your image. If all you need is a count of aggregates, the particle analyzer would help you get here. If you have a z stack, you’ll need the 3D object counter or the 3D Manager.
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