Rapiclear-confocal microscopy

Bella
Bella Posts: 3
in General
Hi
GFP tagging virus was injected in the rat brain and after 3 weeks, rat was purfused.
I sliced brain tissue in 1mm thickness and
cleared with Rapiclear1.47 for several days.
Also, mounted the sample with iSpacer and Rapiclear solution.
However, when I tried to image the sampel with confocal microscopy (olympus) 100x in depth,
focus was not fixed and the result was blurry-greenish image.

I am not sure whether the sample was a problem(not cleared enough) or virus was the problem.
White matter was cleared in a yellowish color but the gray matter was very evident.

Could you share your opinion on this?
Thanks
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Comments

  • SuSam
    SuSam Posts: 6
    I have used RapiClear 1.47 to clear EGFP mouse brain slices in 500um before and the confocal images were pretty sharp. I wonder which objective lens you applied for your image acquisition. I‘d suggest that maybe you can try a glycerin-based immersion lens to acquire images, it worked for me. Hope that will help.
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  • Bella
    Bella Posts: 3
    Thank you very much.
    I think the sample was too thick.
    How did you sliced the sample in 500um?
    Was the sample perfectly cleared just by using the RapiClear 1.47?
    Thank you very much!!
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  • SuSam
    SuSam Posts: 6
    Here’s my protocol for your reference:
    Mouse brain perfused & fixed was sliced to 500um by a vibratome. The slices were fixed with 4% PFA again for 1hr. Wash with PBS 3 times. Transfer samples to 2% PBST overnight at 4°C with gentle shaking. Transfer samples to 0.5% PBST for 1hr at RT with gentle shaking. Wash with PBS 3 times. DAPI staining. Samples immerse in RapiClear 1.47 for 1~2hr at RT with gentle shaking for clearing.
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  • Bella
    Bella Posts: 3
    Thank you very much!!!!
    @SuSam
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