Staining ETC samples

ClarityNL

Has anybody tried to stain samples for imaging yet? I want to try and stain my cleared mouse brain, but don't want to spill my AB's optimizing this. Do any of you have tips regarding staining of thick slides of mouse brain (5 mm slice)?
Cheers, Esther

joelrosiene

@ClarityNL I used a 1:2000 Hoecst stain and a 1:1000 ToPro stain. I modeled my staining off of the original paper, letting the stains sit 25-30 min for thick sections, and 2Hrs for half brains, followed of course by PBS washes. I was able to pick out nuclei when I went to imaging, but I think I had a lot of tissue damage. I had the most success with imaging 800um sections.

JustDave

I'm also curious about results others have gotten doing immunostaining of their cleared samples. I'm trying to label vasculature, nuclei, and macrophages in ETC cleared mouse brain (hemisphere), kidney (half), and liver (nice sized chunk). While I think the clearing worked pretty well I have not been impressed with the quality of staining. My current protocol follows that provided by the authors and includes incubation with primary (at a pretty high concentration) for several days in 1M Na Borate solution at 37C. I wash for about 48 hours in the same buffer and then clear. Although I have seen some staining with IBA1 my attempts with anti-podocin and F4/80 have not seemed to work. Additionally, I incubated samples with 20ug/ml FITC conjugated isolectin to try and label vasculature but this also didn't seem to work on ETC cleared tissue.
Does anyone have any results of staining attempts that they would be willing to share? Has anyone gotten results as well as the authors on very thick tissue pieces or should I try and work with <1mm sections?
Thanks, David

lijun

I did the immunistaing for several times(mouse brain section ,thickness:500um ).But the staining depths always less than 100um.And the nonspecific binding seems quite serious with a high background.My staining protocol are depicted as follow:

The clarified sample(500um) was incubated at 37℃ for 1 day in 0.1%TritonX-100,primary antibody(anti-tublin ,T6793,dilution-1:100)and 1M sodium borate buffer solution ,pH8.5,followed by wash 37℃ for 1day with 0.1%TritonX-100 and 1M sodium borate buffer solution ,pH8.5.
The tissue was then incubated at 37℃ for 1 day in 0.1%TritonX-100,secondary antibody(cy3-goat-antimouse,dilution-1:100)and 1M sodium borate buffer solution ,pH8.5,followed by wash 37℃ for 24h with 0.1%TritonX-100 and 1M sodium borate buffer solution ,pH8.5. And before imaging ,the sample was incubated in FocusClear(300ul) for 24h.

The volume of the diluted staining dye was about 1500ul in a tube(2ml).And when I incubated my sample in the secondary antibody,I also added DAPI in the system(final conc:2ug/ml).But it seemed that the backgound was also too high to recoginzed the nucleus.

Shashank20

Hi all,
I got a huge autofluorescence background both in red and green channel while imaging ETC cleared, and Ab-stained mouse pancreas. To reduce the tissue autofluorescence I even bleached the tissue for 16h in a mixture of MeOH:DMSO:H2O2. The primary and secondary antibodies were diluted in 10% serum in TBST buffer with 5% DMSO. Unfortunatley I could not see any specific staining. Has anybody experienced similar problem with any other mouse tissue ? Pl. share your experience or how to overcome that ? ...Thanks!

patricia

Hi all, I'm just finishing to clear my samples but I'm not sure about the staining process, do you incubate in focusclear in order to achieve the highest transparece of the tissue before staining or after? Thank you!

Kristin_Engberg

Hi Patricia,
You should complete staining after incubating your samples a couple days in PBST. Don't put them in FocusClear until right before you're ready to image (0-1 days).

patricia

Thank you Kristin

patricia

Hi JustDave, I'm curious about your cleared kidney, I have now one into the ETC, could you post an image to see the degree of clearing? Thanks!

JustDave

Patricia,
I'm afraid my attempt failed. I'm going to repeat when I have a chance. This time, I think I'll stick to passive clearing and try a smaller piece of tissue (I tried a kidney half).

patricia

I JustDave, I finally get cleared kidneys, my problem is when staining them. I only get a penetration of about 500um, and couldn't have specific staining of vasculature with lectin. I also tried lectin and B-tubulin in eyes but nothing specific. The only staining that is working for me is DAPI. Does anyone have the same problems of staining specificity?

irina

hey patricia..i am trying to clear eyes for a colleague so i wanted to ask you how do you asses how well they have cleared since they stay black?..i do not know that much about eyes, i have only worked with brains up to now

DavidLenzi

@JustDave, its been a long time since your post, but a recent experiment of mine might be of use to you. I got vascular staining to work in ~1 mm thick sections from an ETC-clarified mouse brain using biotinilated tomato lectin (Vector B1175, 1:100 x 3 days), followed by Dylight 594-streptavidin (Vector SA5594, 1:100 x 1 day), all in the 1M Na-borate buffer from the paper. What IBA1 antibody did you get to work?

helen

I can get staining with a variety of antibodies, but it's a bit hit and miss. I guess i get 200-300um depth routinely, and ~1mm on a good day. Shaking the tissue during incubation seems to make a difference.