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![Confocal microscopic observation of autofluorescence in Adriamycin-induced nephropathy mouse kidney. Images were acquired at excitations of 473 and 559 nm in the fluorescein isothiocyanate (FITC) and Texas Red channels with confocal laser scanning microscopy. Differential interference contrast (DIC) images were acquired at excitation of 473 nm. A, Confocal images of autofluorescence in the frozen kidney section. Arrows represent irregularly shaped granules with high-density fluorescence sprinkled in injured tubule cells. B, Confocal images of autofluorescence in the paraffin-embedded kidney section. Arrows represent red blood cells. C, Confocal images of autofluorescence in the frozen section stained with hematoxylin-eosin (H&E). Arrows represent protein casts (original magnifications 3 200 [A], 3 600 [B], and 3 400 [C]).](profile/Qi-Cao-18/publication/51692969/figure/fig2/AS:277306619777028@1443126458880/Confocal-microscopic-observation-of-autofluorescence-in-Adriamycin-induced-nephropathy.png)
Confocal microscopic observation of autofluorescence in Adriamycin-induced nephropathy mouse kidney. Images were acquired at excitations of 473 and 559 nm in the fluorescein isothiocyanate (FITC) and Texas Red channels with confocal laser scanning microscopy. Differential interference contrast (DIC) images were acquired at excitation of 473 nm. A, Confocal images of autofluorescence in the frozen kidney section. Arrows represent irregularly shaped granules with high-density fluorescence sprinkled in injured tubule cells. B, Confocal images of autofluorescence in the paraffin-embedded kidney section. Arrows represent red blood cells. C, Confocal images of autofluorescence in the frozen section stained with hematoxylin-eosin (H&E). Arrows represent protein casts (original magnifications 3 200 [A], 3 600 [B], and 3 400 [C]).
Source publication
Renal tissue emits intense autofluorescence, making it difficult to differentiate specific immunofluorescence signals and thus limiting its application to clinical biopsy material.
To identify and minimize autofluorescence of renal tissue and demonstrate a simple, efficient method to reduce autofluorescence using Sudan black B.
In this study, the s...
Contexts in source publication
Context 1
... arose from all renal cells in both normal and diseased kidney under FITC and Texas Red channels as shown in Figure 2. Autofluorescence was brighter in tubule cells than in glomeruli. ...
Context 2
... arose from all renal cells in both normal and diseased kidney under FITC and Texas Red channels as shown in Figure 2. Autofluorescence was brighter in tubule cells than in glomeruli. Autofluores- cence was distributed more evenly in normal kidney tissue than in diseased kidney, in which AF showed irregularly shaped granules with high-density fluores- cence sprinkled in kidney cortex (arrows in Figure 2, A). These fluorescent granules were located in inflamed tubulointerstitium and were assumed to be injured tubule and interstitial cells. ...
Context 3
... fluorescent granules were located in inflamed tubulointerstitium and were assumed to be injured tubule and interstitial cells. Some disk-shaped fluorescence in glomeruli and interstitium was identified as arising from red blood cells (arrows in Figure 2, B). Large patches with strong fluorescence were seen in dilated tubules from frozen kidney tissue (arrows in Figure 2, C). ...
Context 4
... disk-shaped fluorescence in glomeruli and interstitium was identified as arising from red blood cells (arrows in Figure 2, B). Large patches with strong fluorescence were seen in dilated tubules from frozen kidney tissue (arrows in Figure 2, C). These bright patches were identified as protein casts using hematoxy- lin-eosin staining. ...
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Citations
... Sodium borohydride (NaBH 4 ) has been reported to reduce AF in neural 9 and respiratory 10 tissues by reducing Schiff bases formed during formaldehyde cross-linking. 11 Sudan Black B (SBB) has been suggested to suppress AF in various types of tissue, including bone marrow/cartilage, 12 neural, 1,3,14 renal, 15 thymus, 16 pancreatic, 17 and intestinal 18 tissues. Glycine has been typically used to quench reactions of free or protein-conjugated formaldehyde in chromatin immunoprecipitation and protein-protein interaction studies. ...
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.
... Sudan Black B (SBB) is a staining dye widely used in histological studies to eliminate autofluorescence, especially in the rat pancreas (4), mouse liver (5) and kidney (6), and the primate brain (7). However, SBB has not been tested in mouse brain tissue in the context of acute brain injury. ...
... These methods mainly include the following strategies to control autofluorescence: (1) Physical masking of the luminescent group of autofluorescence, (2) dissolving and extracting the substance that can produce autofluorescence, and (3) changing the chemical structure of autofluorescence (27). Ammonia-ethanol can extract and dissolve lipids, eliminating lipid-induced autofluorescence (6). Sodium borohydride reduces autofluorescence by destroying fluorophore molecules through oxidation/reduction reactions (28). ...
... SBB is a fat-soluble dye that effectively blocks autofluorescence caused by lipofuscin (25), erythrocyte porphyrins (6), and myeloid granules (4). SBB can block the autofluorescence of tissues without interfering with a particular fluorescence at concentrations of 0.1%-0.5% (3,6,7,29). However, higher concentrations of SBB will mask autofluorescence and specific fluorescence signals, making it unable to obtain A B C FIGURE 4 Sudan black B (SBB) blocks the autofluorescence of brain tissues damaged by traumatic brain injury. ...
Autofluorescence is frequently observed in animal tissues, interfering with an experimental analysis and leading to inaccurate results. Sudan black B (SBB) is a staining dye widely used in histological studies to eliminate autofluorescence. In this study, our objective was to characterize brain tissue autofluorescence present in three models of acute brain injury, including collagenase-induced intracerebral hemorrhage (ICH), traumatic brain injury (TBI), and middle cerebral artery occlusion, and to establish a simple method to block autofluorescence effectively. Using fluorescence microscopy, we examined autofluorescence in brain sections affected by ICH and TBI. In addition, we optimized a protocol to block autofluorescence with SBB pretreatment and evaluated the reduction in fluorescence intensity. Compared to untreated, pretreatment with SBB reduced brain tissue autofluorescence in the ICH model by 73.68% (FITC), 76.05% (Tx Red), and 71.88% (DAPI), respectively. In the TBI model, the ratio of pretreatment to untreated decreased by 56.85% (FITC), 44.28% (Tx Red), and 46.36% (DAPI), respectively. Furthermore, we tested the applicability of the protocol using immunofluorescence staining or Cyanine-5.5 labeling in the three models. SBB treatment is highly effective and can be applied to immunofluorescence and fluorescence label imaging techniques. SBB pretreatment effectively reduced background fluorescence but did not significantly reduce the specific fluorescence signal and greatly improved the signal-to-noise ratio of fluorescence imaging. In conclusion, the optimized SBB pretreatment protocol blocks brain section autofluorescence of the three acute brain injury models.
... Such signal-to-noise ratios may occur when examining tissues which exhibit high amounts of autofluorescence. For instance, murine renal tissues have previously been reported to display intense autofluorescence at a broad range of excitation wavelengths, including those for DAPI, FITC, and Texas Red (Sun et al., 2011;Zhang et al., 2018). Furthermore, fixatives such as aldehydes are known to increase autofluorescence of fixed tissues and impede histological observation (Baschong et al., 2001). ...
Many respiratory pathogens compromise epithelial barrier function during lung infection by disrupting intercellular junctions, such as adherens junctions and tight junctions, that maintain intercellular integrity. This includes Streptococcus pneumoniae, a leading cause of pneumonia, which can successfully breach the epithelial barrier and cause severe infections such as septicemia and meningitis. Fluorescence microscopy analysis on intercellular junction protein manipulation by respiratory pathogens has yielded major advances in our understanding of their pathogenesis. Unfortunately, a lack of automated image analysis tools that can tolerate variability in sample-sample staining has limited the accuracy in evaluating intercellular junction organization quantitatively. We have created an open source, automated Python computer script called “Intercellular Junction Organization Quantification” or IJOQ that can handle a high degree of sample-sample staining variability and robustly measure intercellular junction integrity. In silico validation of IJOQ was successful in analyzing computer generated images containing varying degrees of simulated intercellular junction disruption. Accurate IJOQ analysis was further confirmed using images generated from in vitro and in vivo bacterial infection models. When compared in parallel to a previously published, semi-automated script used to measure intercellular junction organization, IJOQ demonstrated superior analysis for all in vitro and in vivo experiments described herein. These data indicate that IJOQ is an unbiased, easy-to-use tool for fluorescence microscopy analysis and will serve as a valuable, automated resource to rapidly quantify intercellular junction disruption under diverse experimental conditions.
... In brief, slides were rehydrated and antigens were retrieved with citrate buffer (10 mM, pH 6.0) at 95°C for 20 min. Sections were then exposed for 20 min to a 0.1% Sudan Black block diluted in 70% EtOH, as previously, 34 and then blocked further in 5% normal goat serum 0.2% Triton X-100 for 1 h at room temperature. Slides were incubated in humidified chambers overnight at 4°C with the primary antibodies and then the secondary antibodies listed in Table 1. ...
Background
Intraventricular hemorrhage causes significant lifelong mortality and morbidity, especially in preterm born infants. Progress in finding an effective therapy is stymied by a lack of preterm animal models with long-term follow-up. This study addresses this unmet need, using an established model of preterm rabbit IVH and analyzing outcomes out to 1 month of age.
Methods
Rabbit pups were delivered preterm and administered intraperitoneal injection of glycerol at 3 h of life and approximately 58% developed IVH. Neurobehavioral assessment was performed at 1 month of age followed by immunohistochemical labeling of epitopes for neurons, synapses, myelination, and interneurons, analyzed by means of digital quantitation and assessed via two-way ANOVA or Student’s t test.
Results
IVH pups had globally reduced myelin content, an aberrant cortical myelination microstructure, and thinner upper cortical layers (I–III). We also observed a lower number of parvalbumin (PV)-positive interneurons in deeper cortical layers (IV–VI) in IVH animals and reduced numbers of neurons, synapses, and microglia. However, there were no discernable changes in behaviors.
Conclusions
We have established in this preterm pup model that long-term changes after IVH include significant wide-ranging alterations to cortical organization and microstructure. Further work to improve the sensitivity of neurocognitive testing in this species at this age may be required.
Impact
This study uses an established animal model of preterm birth, in which the rabbit pups are truly born preterm, with reduced organ maturation and deprivation of maternally supplied trophic factors.
This is the first study in preterm rabbits that explores the impacts of severe intraventricular hemorrhage beyond 14 days, out to 1 month of age.
Our finding of persisting but subtle global changes including brain white and gray matter will have impact on our understanding of the best path for therapy design and interventions.
... Various histochemical techniques for blocking AF have been evolving. Sudan black B (SBB), trypan blue (TB), copper sulfate (CuSO 4 ), Tris-glycine (Gly), and ammonia/ethanol (AE) have been used to control AF [16][17][18]. The efficacy of chemical reagents in reducing AF differs with the sample type. ...
Immunofluorescence assay is one of methods to understand the spatial biology by visualizing localization of biomolecules in cells and tissues. Autofluorescence, as a common phenomenon in organisms, is a background signal interfering the immunolocalization assay of schistosome biomolecules, and may lead to misinterpretation of the biomolecular function. However, applicable method for reducing the autofluorescence in Schistosoma remains unclear. In order to find a suitable method for reducing autofluorescence of schistosomes, different chemical reagents, such as Sudan black B (SBB), trypan blue (TB), copper sulfate (CuSO4), Tris-glycine (Gly), and ammonia/ethanol (AE), at different concentrations and treatment time were tested, and SBB and CuSO4 were verified for the effect of blocking autofluorescence in immunofluorescence to localize the target with anti-SjCRT antibody. By comparing the autofluorescence characteristics of different conditions, it was found that SBB, TB and CuSO4 had a certain degree of reducing autofluorescence effect, and the best effect in females was using 50 mM CuSO4 for 6 h and in males was 0.5% SBB for 6 h. Furthermore, we have applied the optimized conditions to the immunofluorescence of SjCRT protein, and the results revealed that the immunofluorescence signal of SjCRT was clearly visible without autofluorescence interference. We present an effective method to reduce autofluorescence in male and female worm of Schistosoma japonicum for immunofluorescence assay, which could be helpful to better understand biomolecular functions. Our method provides an idea for immunofluorescence assay in other flukes with autofluoresence.
... Tubule segments marker AQP1 indicates the proximal tubule cells (b), AQP2 indicates the cortical and medullary collecting duct (e) and PNA indicates the distal tubule cells (h). Renin can be clearly seen in the juxtaglomerular apparatus (JGA, *indicates glomeruli) and blood vessels (#) clearly distinguishable from the low level autofluorescence in the renal tubules consistent with that previously described58 . Arrows indicate positive staining and scale bar = 100 µm for all images. ...
We examined the effect of total and afferent renal denervation (RDN) on hypertension and the renin-angiotensin system (RAS) in a rodent model of juvenile-onset polycystic kidney disease (PKD). Lewis Polycystic Kidney (LPK) and control rats received total, afferent or sham RDN by periaxonal application of phenol, capsaicin or normal saline, respectively, and were monitored for 4-weeks. Afferent RDN did not affect systolic blood pressure (SBP) determined by radiotelemetry in either strain (n = 19) while total RDN significantly reduced SBP in Lewis rats 4-weeks post-denervation (total vs. sham, 122 ± 1 vs. 130 ± 2 mmHg, P = 0.002, n = 25). Plasma and kidney renin content determined by radioimmunoassay were significantly lower in LPK vs. Lewis (plasma: 278.2 ± 6.7 vs. 376.5 ± 11.9 ng Ang I/ml/h; kidney: 260.1 ± 6.3 vs. 753.2 ± 37.9 ng Ang I/mg/h, P < 0.001, n = 26). These parameters were not affected by RDN. Intrarenal mRNA expression levels of renin, angiotensinogen, angiotensin-converting enzyme (ACE)2, and angiotensin II receptor type 1a were significantly lower, whereas ACE1 expression was significantly higher in the LPK vs. Lewis (all P < 0.05, n = 26). This pattern of intrarenal RAS expression was not changed by RDN. In conclusion, RDN does not affect hypertension or the RAS in the LPK model and indicates RDN might not be a suitable antihypertensive strategy for individuals with juvenile-onset PKD.
... With respect to applying super-resolution microscopy for diagnostic purposes, tissue samples are more likely to originate from elderly patients and thus autofluorescence might become a major issue. It has been observed in numerous studies on human [51,52] as well as murine FFPE kidney tissue and its full reduction proved challenging [47,53,54]. Therefore, if a method other than SMLM is used, we suggest not to invest in reducing the background due to its negligible effect on kidney tissue autofluorescence. ...
Immunofluorescence microscopy is routinely used in the diagnosis of and research on renal impairments. However, this highly specific technique is restricted in its maximum resolution to about 250 nm in the lateral and 700 nm in the axial directions and thus not sufficient to investigate the fine subcellular structure of the kidney’s glomerular filtration barrier. In contrast, electron microscopy offers high resolution, but this comes at the cost of poor preservation of immunogenic epitopes and antibody penetration alongside a low throughput. Many of these drawbacks were overcome with the advent of super-resolution microscopy methods. So far, four different super-resolution approaches have been used to study the kidney: single-molecule localization microscopy (SMLM), stimulated emission depletion (STED) microscopy, structured illumination microscopy (SIM), and expansion microscopy (ExM), however, using different preservation methods and widely varying labelling strategies. In this work, all four methods were applied and critically compared on kidney slices obtained from samples treated with the most commonly used preservation technique: fixation by formalin and embedding in paraffin (FFPE). Strengths and weaknesses, as well as the practicalities of each method, are discussed to enable users of super-resolution microscopy in renal research make an informed decision on the best choice of technique. The methods discussed enable the efficient investigation of biopsies stored in kidney banks around the world.
Graphical abstract
... In line with the recommendation of others, we found that the use of copper sulfate with ammonium chloride in the final rinsing step helped in quenching the autofluorescence effect frequently seen in paraffin-embedded tissues (Schnell et al., 1999). Other protocols described the use of Sudan Black B in ethanol or other reagents to reduce the autofluorescence signals (Sun et al., 2011;Gandhi and Khare, 2018). However, Sudan Black B is incompatible with some xylene-based permanent mounting media (Schnell et al., 1999), and it reduces both specific and non-specific signal intensities drastically (Gandhi and Khare, 2018). ...
Immunofluorescence staining is the most frequently applied technique to detect and
visualize various molecules in biological samples. Many protocols can be found in the
literature and the websites of commercial antibody producers. This can result in a time consuming and costly methodical work to establish “simple” antibody staining. We
here summarize in a stepwise manner an easy-to-follow immunofluorescence staining
protocol with an improved specific fluorescent signal and a reduced background and
non-specific binding signal. This will help scientists to save time, effort, and antibody
costs during the application of such a valuable technique.
... This is important for distinguishing true signal from background signal during microscopy since the bladder epithelium is auto-fluorescent in multiple channels. In addition to the scramble probe, blocking with 0.1% Sudan Black B prior to mounting may reduce background autofluorescence inherent in the tissue 20 . ...
Visualization of the interaction of bacteria with host mucosal surfaces and tissues can provide valuable insight into mechanisms of pathogenesis. While visualization of bacterial pathogens in animal models of infection can rely on bacterial strains engineered to express fluorescent proteins such as GFP, visualization of bacteria within the mucosa of biopsies or tissue obtained from human patients requires an unbiased method. Here, we describe an efficient method for the detection of tissue-associated bacteria in human biopsy sections. This method utilizes fluorescent in situ hybridization (FISH) with a fluorescently labeled universal oligonucleotide probe for 16S rRNA to label tissue-associated bacteria within bladder biopsy sections acquired from patients suffering from recurrent urinary tract infection. Through use of a universal 16S rRNA probe, bacteria can be detected without prior knowledge of species, genera, or biochemical characteristics, such as lipopolysaccharide (LPS), that would be required for detection by immunofluorescence experiments. We describe a complete protocol for 16S rRNA FISH from biopsy fixation to imaging by confocal microscopy. This protocol can be adapted for use in almost any type of tissue and represents a powerful tool for the unbiased visualization of clinically-relevant bacterial-host interactions in patient tissue. Furthermore, using species or genera-specific probes, this protocol can be adapted for the detection of specific bacterial pathogens within patient tissue.
... Autofluorescence, either intrinsic or induced by fixation and tissue processing, may either mask specific fluorescent signals or be mistaken for fluorescent labels [67]. Autofluorescence interferes with routine specific fluorescent labeling by red, green, and blue fluorescent probes, making it very difficult to visualize or co-localize multiple proteins of interest [68]. Baschong et al. [67] have listed three different strategies can be used to control autofluorescence: extraction of the autofluorescent constituent by dissolution (ammonia-ethanol), chemical modification of the fluorochrome (borohydride), and masking of the autofluorescent structure by appropriate staining (Sudan Black). ...
