Management of autofluorescence in formaldehyde-fixed myocardium: choosing the right treatment

Abstract

Autofluorescence (AF) poses challenges for detecting proteins of interest in situ when employing immunofluorescence (IF) microscopy. This interference is particularly pronounced in strongly autofluorescent tissues such as myocardium, where tissue AF can be comparable to IF. Although various histochemical methods have been developed to achieve effective AF suppression in different types of tissue, their applications on myocardial samples have not been well validated. Due to inconsistency across different autofluorescent structures in sometypes of tissue, it is unclear if these methods can effectively suppress AF across all autofluorescent structures within the myocardium. Here, we quantitatively evaluated the performance of several commonly used quenching treatments on formaldehyde-fixed myocardial samples, including 0.3 M glycine, 0.3% Sudan Black B (SBB), 0.1% and 1% sodium borohydride (NaBH4), TrueVIEW® and TrueBlack®. We further assessed their quenching performance by employing the pre-treatment and post-treatment protocols, designed to cover two common IF staining scenarios where buffers contained detergents or not. The results suggest that SBB and TrueBlack® outperform other reagents in AF suppression on formaldehyde-fixed myocardial samples in both protocols. Furthermore, we inspected the quenching performance of SBB and TrueBlack® on major autofluorescent myocardial structures and evaluated their influence on IF imaging. The results suggest that SBB outperforms TrueBlack® in quenching major autofluorescent structures, while TrueBlack® excels in preserving IF labeling signal. Surprisingly, we found the treatment of NaBH4 increased AF signal and enhanced the AF contrast of major autofluorescent structures. This finding suggests that NaBH4 has the potential to act as an AF enhancer and may facilitate the interpretation of myocardial structures without the need for counterstaining.

Quantification of cardiac capillarization in basement-membrane-immunostained myocardial slices using Segment Anything Model

“…4 a2,b2), autofluorescent features (Fig. 4 a1,b1) as fiducial features are provided 26 . SAM-Only demonstrated inaccurate segmentation, especially for small objects, and ineffective classification.…”

In Situ Structural Characterization of Cardiomyocyte Microenvironment by Multimodal STED Microscopy

“…SHG imaging detects intrinsic SHG sources in the myocardium in a label-free manner, providing an elegant approach for the background-free localization of myocardial structures. Tissue AF, arising from endogenous fluorophores and fixative-induced fluorescence, can also be used to facilitate the interpretation of myocardial structures in tissue samples [25]. AF imaging can be conducted utilizing the same setup as confocal IF imaging.…”

“…Thus, AF imaging not only inherits an optical-sectioning capability but also intrinsically complements IF imaging. We have identified autofluorescent structures in cardiomyocytes as mitochondria, while the endothelium of microvessels and the endocardium also produce observable AF contrast [25]. To demonstrate 3D multiplexed imaging utilizing the contrast mechanisms of IF, AF, and SHG, β1 integrin (transmembrane protein) or collagen type III (ECM structural protein) in mouse myocardial sections were IF stained.…”

“…Due to the diffraction-limited resolution, the perimysial layer cannot be separated from the endocardium. Fortunately, some clues can be found using endothelial cell nuclei as a reference, as nuclei have no AF contrast [25] (see also Figure S1). The absence of collagen type III on the surface of two endothelial cells (Figure 5, arrows in top row) provides direct evidence that the endocardium forms an outer layer enclosing the perimysium of the papillary muscle.…”

Protocol refinement for quenching autofluorescence of red blood cells in FFPE sections of organ samples from cattle, pigs and chickens

“…The effectiveness of incubating sections in Sudan Black B has been previously shown [14][15][16][17], including the use of mere DMSO as a solvent [18]; TrueBlack® in DMSO, Biotium) however, not using DMSO as an additional solvent to ethanol. In addition, such protocol aspects have not been thoroughly studied in tissue sections from chickens, cattle and pigs.…”