Rapiclear-confocal microscopy

Bella

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

SuSam

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.

Bella

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!!

SuSam

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.

Bella

Thank you very much!!!!
@SuSam