Animal experiments and molecular/cellular biology experiments have long relied on fluorescence microscopy. But there's usually lots of autofluorescence in tissues, messing up the image and yielding false results. A lack of controls can make false-positive detections and therefore wrong detections. Broad and intense autofluorescence was detected in brain tissues destroyed by ischemic stroke and hemorrhagic stroke.

Sudan Black B (SBB) is a staining dye widely used in histologic studies to eliminate autofluorescence. Studies have shown that SBB treatment is effective in reducing background autofluorescence. In addition, SBB treatment has shown excellent applicability in fluorescent labeling protocols and immunofluorescent staining of intracerebral hemorrhage (ICH) frozen brain sections. In addition, it has an effective blocking effect on the autofluorescence of brain tissue in paraffin-embedded brain sections with cerebral ischemic injury.

Overview of SBB

SBB primarily works by physically masking autofluorescence through adsorption, which enhances its light absorption capabilities. This treatment effectively blocks emission peaks from damaged tissue components that produce high autofluorescence, resulting in a more homogeneous background and improved visibility of specific fluorescence signals.

Advantages of SBB

• SBB dye can block the emission peaks of damaged tissue components that produce prominent high autofluorescence.
• SBB dye reduces the overall autofluorescence, giving brain tissue a homogeneous background and highlighting specific fluorescence.
• SBB masks autofluorescent structures rather than physicochemical binding, effectively reducing the autofluorescence signal without interfering with specific fluorescence signals.

Disadvantages of SBB

• SBB can significantly improve the signal-to-noise ratio of fluorescence imaging, but the physical masking effect of SBB reduces the total fluorescence signal to some extent.
• SBB processing adds a processing step that requires a specific incubation time to reduce tissue autofluorescence.

Autofluorescence in Brain Tissues Damaged by ICH

The researchers fluoresced the striatum of collagenase-induced ICH model injury with fluorescence microscopy using fluorescein isothiocyanate (FITC), Texas red (Tx Red), and 4', 6-diamidino-2-phenylindole filters (DAPI) filters. They plotted the spatial distribution of autofluorescence by adding a horizontal axis to the photograph of the site of the haemorrhage and dividing the fluorescence along that axis. The results showed that fluorescence intensity was low away from the hemorrhage site, increased significantly around the hemorrhage site, and was extremely low inside the hemorrhage site, then increased again around the hemorrhage site. Overall, the autofluorescence of ICH-injured brain tissue mainly originated from the tissue around the hemorrhagic foci, and its intensity was significantly higher than that of other surrounding regions.

Fig.1. Characteristics and distribution of autofluorescence in brain tissues damaged by ICH. (Wang, et al., 2023)

Causes of Autofluorescence in Brain Tissues Damaged by ICH

ICH leads to significant erythrocyte leakage, with degradation products generating protoporphyrin, a likely contributor to autofluorescence near hemorrhagic foci. Additionally, it mentions that lipofuscin and tissue necrosis also produce considerable autofluorescence, complicating fluorescence imaging.

SBB Blocks Autofluorescence of Brain Tissues Damaged by ICH

By observing the autofluorescence intensity of the same brain region (striatum) in the same brain section of ICH injury before and after SBB treatment, it was found that there was a strong non-specific fluorescence signal in the untreated samples, and SBB treatment significantly reduced the autofluorescence on all the filters.

In addition, the study examined the fluorescence signal of IL-10 labeled with Cy5.5 and found that SBB treatment significantly increased its visibility, resulting in a decrease in background autofluorescence. For neuronal evaluation, NeuN was labeled with fluorescent labeling, and the SBB treatment not only reduced the background signal but also enhanced specific fluorescence, resulting in a significant decrease in autofluorescence.

Fig.2. SBB blocks autofluorescence of brain tissues damaged by ICH. (Wang, et al., 2023)

Comparison of Immunofluorescence of Brain Tissues Damaged by Cerebral Ischemia Before and After SBB Treatment

The effect of SBB on paraffin-embedded brain sections was verified by immunofluorescence staining of ischemic injured brain tissue. Compared with the untreated control group, SBB treatment significantly reduced the background fluorescence and highlighted the specific immunofluorescence signal of the injured brain tissue in paraffin-embedded sections.

Fig.3. SBB blocks autofluorescence of brain tissues damaged by cerebral ischemia in a middle cerebral artery occlusion (MCAO) model. (Wang, et al., 2023)

1. Wang, S., et al. (2023). Blocking autofluorescence in brain tissues affected by ischemic stroke, hemorrhagic stroke, or traumatic brain injury. Frontiers in Immunology, 14, 1168292.