Hi @Tomo . Can you tell us the exact procedure/parameters of your ETC. Are you using Focus Clear or something else for refractive index matching? Did the brain look clear? Was there any yellowish color? Did you try sectioning the ETC cleared brain to check for the signal?
Hi @raju, I sincerely appreciate your help to troubleshoot our issue, hoping this helps others who has similar issues.
ETC parameter; I tested 20V at 37C for 48 hours as well as 60V at 50C for 48 hours. Embedding media; we used 80% glycerol. Clearing result; yes, looked cleared, no yellowish color at all Sectioning the ETC cleared tissue (vibratome?); no, I didn't mainly because I didn't see any epi-fluorescence from the samples. Confocal; Olympus FV1000
Although our organs of interest are lung, kidney, adipose tissue, intestine and skin, we included brain from transgenic where all cells expressing membrane-bound TdTomato as a positive control for the method. Again, cyro sectioning of the tissue had a strong signal of Tomato.
Hi @Tomo, did I get it right that you are using Glycerol in place of Focus Clear for refractive index matching and mounting? One thing with Glycerol is that the fluorophores tend to bleach very fast in it. You may consider adding anti-fading agent (eg, I would recommend DABCO 2.5 mg/ml). [My suspicion is that you might be already loosing your fluorescence in glycerol clearing. How long do you clear in it?]
I will suggest that you also test out with sections i.e. after embedding the tissue in hydrogel, slice it into 1 mm thick slice, put it in cleaning SDS/Boric Acid clearing solution @37 C with shaking for a couple of days (passive clearing), wash off the SDS with PBS for a day. Check out in Confocal in PBS itself (tissue wont be clear but you should be able to see if there is any signal on the surface). Put the slice in Focus Clear (if available and preferred), otherwise 87% glycerol with DABCO (2.5 mg/ml) (or any other anti-fading agent) in it. Check out in Confocal again. Lemme know. tomerr at stanford.
raju and others, thanks for all the helpful info. im getting closer to imaging again with cleared brains that i have stained with antibodies. quick question: how long do you leave your samples in focus clear before you imaging? ive read a couple of different posts concerning the time. thanks.
@rspence before imaging ,I usually immersed my brain section(500um) in FocusClear for 1day. And I observed that although the sample looks like quite transparent with my naked eye,the intensity of antibody decrease as I increase the imaging depth in the z direction.
So I have been working with passively cleared tissue and been having the same autofluorescence issue that everyone seems to be having. Confocal clears it up a bit and 2-photon looks even nicer.. I have been thinking that this may be caused by remaining SDS in the tissue. From what I have been told, SDS can cause significant autofluorescence issues. I am curious if anyone has tried dialysis to remove more of the SDS from the tissue. Fabio mentioned that he saw AF after the hydrogel step, so this may be just a stab in the dark.
sorry to put this on again. i have similar problems as most of you mentioned. i passive clear brain samples ( whole brains and also 1 mm thick slices). The samples look completely cleared in the clearing solution. Once i put them into PBS they appear more turbid, but after putting them into RIMS for 1 day they look fine. I place them in a Willco dish and fill in some of the RIMS ( i dont use a sealed chamber) and use a inverted confocal miscroscope. I am not able to detect anything beside heavy autofluorescence in all channels.
well using the lamp you will only see autofluorescence. Sometimes if your signal is really strong you are able to see it. For me the best option is to use the binocular to search for an area in the tissue where I know there should be signal, then change to confocal mode and look for it.
changing to confocal microscopy helped me a lot. with fluorescent light i couldn'tsee anything beside autofluorescence. i could only see antibody stained cells, endogenous and viral GFP were gone. has anybody experienced the same?
Comments
ETC parameter; I tested 20V at 37C for 48 hours as well as 60V at 50C for 48 hours.
Embedding media; we used 80% glycerol.
Clearing result; yes, looked cleared, no yellowish color at all
Sectioning the ETC cleared tissue (vibratome?); no, I didn't mainly because I didn't see any epi-fluorescence from the samples.
Confocal; Olympus FV1000
Although our organs of interest are lung, kidney, adipose tissue, intestine and skin, we included brain from transgenic where all cells expressing membrane-bound TdTomato as a positive control for the method. Again, cyro sectioning of the tissue had a strong signal of Tomato.
I will suggest that you also test out with sections i.e. after embedding the tissue in hydrogel, slice it into 1 mm thick slice, put it in cleaning SDS/Boric Acid clearing solution @37 C with shaking for a couple of days (passive clearing), wash off the SDS with PBS for a day. Check out in Confocal in PBS itself (tissue wont be clear but you should be able to see if there is any signal on the surface). Put the slice in Focus Clear (if available and preferred), otherwise 87% glycerol with DABCO (2.5 mg/ml) (or any other anti-fading agent) in it. Check out in Confocal again.
Lemme know. tomerr at stanford.
Are you looking in the confocal mode? or just using the UV lamp?
I use brain samples so is easier for me i guess.