If sudan black does not work, copper sulfate in ammonium acetate buffer (pH 5) should do the trick. If covers too much of fluorescent dye just reduce incubation time.

Here is link to a paper explaining the method: http://journals.sagepub.com/doi/full/10.1177/002215549904700601

Best regards

Dear Zjalic,

thanks for the advise. I´ll try it asap and let you know the outcome.

Best

Felix

Dear Felix,

just for my information: have you fixated the tissue? Some fixatives do result in high autofluorescence. Kind regards, Harrie Verhoeven.

Dear Harrie, 

I perfused with 4% PFA (in h2o) and post-fixed for 2h on ice. Do you recommend something different?

The strange thing is, that this autofluorescence appears in different regions of the mouse brain depending on the laser I use for excitation. If I use the 488nm, the striatum shines brightly (which I dont really care about for my analysis). If I use the 633nm, the cc does - and thats a problem for my analysis. 

Today I'll take a look on how much the different 2nd antibodys contribute to this effect. 

Best

Felix

For the visualization what this question is about, please see the attached file:

Pictures on the left side: staining without a 647nm-Fluorochrome antibody but the cc still shines brighter than the cortex when ectited with a 633nm laser.

Pictures on the right side: staining of nuclear Olig2 (yellow arrowheads in Cx and green arrows in cc) and application of a 594nm-Fluorochrome antibody as well as 647nm-Fluorochrome antibody. Olig2 positiv signals in the far-red channel (grey) are "lost" in the cc due to high autofluorescence.

Blue dotted line seperated Cx from cc.

These brainslices are 9µm thin frozen sections and were treated with 0,3% SudanBlack for 7min after 2nd antibody incubation.

Best

Felix

Dear Felix,

Very nice pictures! PFA  has some issues about autofluorescence, especially in brain sections. Maybe you could use this technique:

https://www.ncbi.nlm.nih.gov/pubmed/28892031

At the moment, I am constructing a device for driving a 40W white led for microscopy illumination. It may well suit your problem as well.

I hope this helps improving your already impressive results. Kind regards, Harrie Verhoeven

Dear Harrie,

funny thing is, I have thought about a "pre-photobleaching approach" about a year ago but thought the idea is too fancy. Really cool to see, that there is actually a paper about it!  Thanks a lot.

(just noticed, that I don´t have access to the PDF - would you mind sending it to me, please?)

Best regards,

Felix

Dear Felix, I will send it to later this evening.

Kind regards, Harrie

Felix,

I have to track back on my promise to send you the file. I do not have access to the University network at the moment. I will try later.

Felix,

another paper which is accessible:

https://doi.org/10.1016/j.jneumeth.2013.08.018

Kind regards, Harrie

Thank you again, Harrie!

There´s just one problem to it. Since I inject GFP-positive cells into the mouse brain, they may get bleached as well. One would have to try to use antibodys for the detection of GFP following this LED-technique.

It´ll be my pleasure to keep you and everyone interested updated on that matter.

Best regards,

Felix

Dear Felix,

in this case I would like to suggest to use some of the LED high power modules that have been developed for horticultural purposes. They are now close to 80W for a four chip device, with 420 nm, 520 nm, 630nm and a white light chip. The spectral distributions of each chip are well defined, By selecting the red and green chip, you can bleach all autofluorescence above 500 nm, without affecting the GFP performance, since the excitation spectrum of GFP is way below 520 nm. Have a look at this device: CLP6C-FKB-CKNPRGJBB7A363. Data are available at Mouser.com. Kind regards, Harrie Verhoeven.