sudan black b treatment

[funk.106]

Hi all! So I am trying to do a fluorescent colocalization study on methacarn-fixed paraffin-embedded human hippocampal tissue from Alzheimer's disease patients, meaning I've got some nasty autofluorescence going on. To combat this, I've started using 0.1% Sudan Black B dissolved in 80% ethanol at the end of the protocol (after washing the secondary ab) to try to darken the autofluorescence, and this seems to be helping the autofluorescence, but as always it's creating a new problem. I'm finding that it seems to be making my slides blurry when I look at them using a confocal microscope. Does anyone have any insight into why this may be happening? The picture was really crisp before starting the SB treatment, so it's not the antibodies. I'm leaning towards blaming the ethanol, so I'm going to try dissolving it in water and filtering it instead, but I thought I'd ask you guys about it also. Thanks!!

[LoudTrout]

did the water work out ??

[funk.106]

Well, no....Sudan Black B is nearly impossible to dissolve in water. What I found that worked for me was I dissolve it in 70% ethanol and filter it. I apply the SBB to the tissue for 8 minutes. I then wash twice in PBST for 10 minutes each and then 10 minutes in phosphate buffer (no tween).