Reduction of autofluorescence in whole adult worms of Schistosoma japonicum for immunofluorescence assay

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.

AF, either intrinsic or induced by fixation processing, may either mask specific fluorescence signals or be mistaken for fluorescent labels [7].
Biological AF, emitted from endogenous fluorophores, is an intrinsic property of cells and tissues, commonly seen in mammals [8,9], rodents [10], nematodes [11], and Schistosoma [12]. It is also noted that the AF properties of specific tissue constituents may be of diagnostic value or indicative of cell viability. In schistosomes, the AF of eggs has been used to detect eggs in diseased tissues [13]. AF of the vitelline gland in female schistosomes is emitted mainly from vitelline cells, which can be used to separate and enrich vitelline cells [14] or applied for vitelline gland localization [15]. However, AF is often a noise signal in IFA, and therefore has been a significant concern.
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. It was reported that CuSO 4 was used to quench AF within the vitellarium of Schistosoma mansoni [19], but little is known of applicable reagents for reducing the AF in S. japonicum.
The present study characterized the AF of male and female S. japonicum, and tested five chemical reagents to assess their efficacy in reducing the AF in IFA.
Parasites and animals were prepared. Female Kunming mice (20-25 g) (Shanghai Animal Center, Chinese Academy of Sciences, China) were infected with 80 ± 5 S. japonicum cercariae (provided by the National Institute of Parasitic Diseases, China Center for Disease Control and Prevention). Adult worms were harvested by perfusion with ice-cold 0.9% NaCl solution containing heparin (10 U/mL) (Sangon Bioengineering Technical Services, China) at 28 days post-infection. Male and female worms were gently separated, fixed with 4% paraformaldehyde (Sangon Bioengineering Technical Services, China) for 2 h at room temperature, and then kept overnight at 4 °C.
To ascertain the effectiveness of different reagents in controlling the AF arising from schistosomes, we tested five chemical reagents (Sigma-Aldrich), including CuSO 4 , SBB, TB, Gly, and AE, at different concentrations and for different treatment times. The whole worms were immersed in CuSO 4 at 0.5, 5, or 50 mM in 50 mM ammonia acetate for 1.5, 3, or 6 h; in 0.01%, 0.1%, or 0.5% SBB in 70% ethanol for 1, 2, or 6 h; with 0.05% TB for 1 or 2 h; with 0.1 M Gly in TBS (pH 7.4) for 2 h; and in 0.25% ammonia in 70% ethanol for 2 h. All procedures were performed at room temperature. To remove the excess testing regents, the worms were washed six times for 20 min each with 0.02% Tween 20 in PBS (PBST). The worms were then placed on a slide, mounted with 80% glycerol, and viewed with CLSM.
To verify the reactivity of the anti-SjCRT antibodies with crude antigens of S. japonicum worms, western blotting (WB) was performed. The worm protein was extracted with 10-20 worms in 1 ml PBS by sonication on ice and then centrifuged for 10 min at 13,000×g at 4 °C. WB was performed as described previously [20]. The protein extracts (50 μg protein) of adult female and male worms were resolved by 12% SDS-polyacrylamide gel electrophoresis (PAGE) and electrotransferred onto a polyvinylidene fluoride membrane. The membrane was IFA was performed as described in a previous report [15]. After fixation, blocking of non-specific antigens, and treatment with the optimal conditions to control AF, the worms were incubated with rabbit anti-SjCRT IgG (1:300-500) in blocking solution at 4 °C for 3 days, followed by washing three times for 2 h each with PBST. For fluorescence staining, the worms were incubated with Cy3-labeled goat anti-rabbit IgG (1:500) in blocking solution at 4 °C for 3 days, and at the end of the second day, DAPI staining solution (1:50) was added to the incubation solution. After washing three times for 2 h each with PBST, the worms were placed on a slide, mounted with 80% glycerol, and viewed using CLSM (Nikon A1R, Nikon Instruments Inc., Japan). In IFA, DAPI and Cy3 fluorescent dyes were used for labeling nuclear and SjCRT protein, respectively [21,22], and the fluorescence signals were detected in DAPI and Cy3 channels, respectively. The EGFP detection channel was used for monitoring green AF and evaluating the AF reduction effect. The fluorescence images of whole-mount worms were examined using CLSM. The DAPI, Cy3, and EGFP channels of microscopy conditions for fluorescence imaging are shown in Table 1. The DAPI was purchased from Boster Biological Technology, China. The Cy3-labeled goat anti-rabbit IgG was from Beyotime Biotechnology, China.
After preparative processing, the worms were mounted with 80% glycerol and placed under a confocal laser microscope for observation. It was observed that both female and male worms had AF in four different channels (DAPI, EGFP, mCherry, and AF647). In female worms, AF was seen in the tegument, vitelline gland, ovary, eggs, and the worm head (Fig. 1a). However, no blue AF was observed in the vitelline gland, ovary, or eggs ( Fig. 1a DAPI column). AF in male schistosomes was mainly distributed in the intestine, tegument, and gynecophoric canal (Fig. 1b). In males, the fluorescence intensity at the intestine was stronger. In male and female worms, the strongest intensity of AF was seen under the EGFP channel compared to others (Fig. 1).
Five different chemical reagents were used to treat male and female worms for reduction of AF, and it was found that the reduction effect varied among reagents, with Gly and AE having no obvious effects (Additional file 1: Figure S1), while CuSO 4 , SBB, and TB had some effect (Fig. 2). It was noted that the effect of blocking AF also differed with the worm sex and the duration of chemical exposure. After treatment with CuSO 4 , the AF in both male and female worms was reduced, although the reduction was more significant in female worms ( Fig. 2b and c). In females treated with 5 mM CuSO 4 for 1.5 h, the AF was attenuated in all four tested channels (Fig. 2b), while treatment for 3 h led to no detectable AF in mCherry and AF647 channels (Fig. 2c). There was no significant reduction in AF in the four channels in males treated with 5 mM of CuSO 4 for 1.5 h (Fig. 2b), whereas a reduction was seen in mCherry and AF647 channels after 3 h of treatment (Fig. 2c).
SBB exhibited some effect in controlling AF in both males and females, particularly in males ( Fig. 2d and e). In females treated with 0.1% SBB for 2 h, AF was reduced in the mCherry channel and blocked in the AF647 channel (Fig. 2e), but treatment with 0.1% SBB for 1 h did not exert a perceptible AF reduction effect in females (Fig. 2d). Interestingly, incubation with 0.1% SBB for 1 h significantly attenuated AF in male worms in the EGFP and mCherry channels (Fig. 2d); the AF intensity in males in DAPI and EGFP channels was significantly reduced after treatment for 2 h, while it was blocked in the mCherry and AF647 channels (Fig. 2e). TB showed certain reductive effects on AF in male and female worms, but only in the AF647 channel ( Fig. 2f  and g).
It was demonstrated that CuSO 4 presented better reduction effects on AF in female worms and SBB in male worms, and longer time exposure to reagents enhanced the reduction of AF. Different concentrations of CuSO 4 and SBB were used to treat worms, and the results are shown in Fig. 3. Compared with the control, all the defined concentrations of CuSO 4 and SBB had AF reduction effects. The AF reduction effects in female worms were increased with increasing CuSO 4 concentration, with the AF detected in the EGFP channel more difficult to remove than that in other channels. The effect of AF in male worms was not significantly different between 0.01% SBB and 0.1% SBB, but the effect of 0.5% SBB treatment was significantly better. Among all the chemical solutions, 50 mM CuSO 4 showed the best AF reduction effect in female worms (Fig. 3a), while 0.5% SBB had the best AF reduction effect in male worms (Fig. 3b).
WB was used to verify the reactivity of the anti-SjCRT antibodies with crude antigens of male and female worms. The results showed a single band in the crude antigen lanes of both male and female worms, with molecular weight of around 55 kDa (Additional file 2: Figure S2). The band of the female protein lane was less intense than that of the male protein lane (Additional file 2: Figure S2).
In order to verify the effect of AF reduction conditions applied in immunofluorescence experiments, we performed immunofluorescence localization assay on SjCRT. In this assay, fluorescent dye Cy3-labeled goat anti-rabbit IgG was used to localize SjCRT; the nuclei were stained by DAPI. In addition to setting up a normal rabbit serum control, we also monitored the AF using EGFP channels unoccupied by any fluorescent dye. With the treatment for AF reduction in IFA, no significant AF signal was seen in either females or males under the EGFP channel ( Fig. 4a and b, Additional file 3: Figure  S3). In the normal rabbit serum control, no non-specific immunofluorescence signal was detected in females, but a weak non-specific immunofluorescence signal was seen in males under the Cy3 channel (Additional file 3: Figure  S3). The nuclear staining and SjCRT IF staining signals were scattered and granular in both females and males (Fig. 4), and the weak non-specific immunofluorescence  Table 1. Scale bar: 500 μm signal did not interfere with the SjCRT signal in males. The IF signals of SjCRT were seen in the anterior end of the esophagus and tegument of female worms (i and ii in Fig. 4c), and in the ventral sucker and dorsal tegument of male worms (iii and iv in Fig. 4c).
Previous studies have shown that both male and female worms of S. japonicum can produce AF under different excitation light irradiation (405, 458, 476, 488, 514, 543, and 633 nm), with the strongest intensity for green AF (488 nm excitation) [12]. Our results (Fig. 1) show that AF could be observed in S. japonicum worms at 405, 488, 561, and 640 nm excitation wavelengths, with the highest AF intensity produced with excitation at a wavelength of 488 nm. It was also shown that the AF of the male and female tegument and the male intestine was detectable in all channels (Fig. 1), but no AF was detected under the DAPI channel in the vitelline glands or eggs of females (Fig. 1a), which may be associated with endogenous fluorescent substances in the vitelline glands and eggs. Common endogenous fluorescent substances in organisms include amino acids, structural proteins, vitamins, lipopigments, flavins, and porphyrins [23]. During the development of the vitelline gland of schistosome females, three types of inclusions accumulate in the vitelline cells: shell globule clusters, lipid droplets, and β-glycogen particles [24]. However, the co-localization results for AF and lipid droplet staining of vitelline cells showed that AF did not derive from lipid droplets [14].
The AF features of schistosomes have been applied for diagnosis and vitelline cell separation [13,14], but AF is mainly an interfering signal in IFA for schistosome molecules. In previous studies, IFA of schistosome molecular localization showed that a stronger green AF noise signal was seen in the female gonad and cecum [25] and the eggshell [26,27], and a weaker red AF signal was observed in female adult schistosomes [28,29]. In our results, AF was detected in female and male worms in four different channels (Fig. 1). All cases of AF mentioned above suggest that AF reduction is important for the IFA of schistosome molecules. Our assays revealed that CuSO4, SBB, and TB had various effects (Fig. 2) on AF reduction in S. japonicum: 50 mM CuSO4 reduced AF in females and 0.5% SBB reduced AF in males (Fig. 3). CuSO 4 and SBB are widely used chemical reagents to remove AF. In fact, the AF reduction effect of both CuSO 4 and SBB can be seen in samples with lipofuscin-like AF [30]. Previous studies have applied CuSO 4 to reduce AF of schistosomes for fluorescence in situ hybridization (FISH) [19]. In our study, both CuSO 4 and SBB were found to be effective in AF reduction for both female and male worms. However, CuSO 4 was more effective for females, while SBB was more effective for males (Fig. 2), although the underlying mechanism of CuSO 4 and SBB in AF reduction is not well understood. This difference in AF reduction between males and females still suggests that the reasons for the formation of AF in females and males may not be exactly the same. Triglycerides (TG) have been reported as a lipid associated with AF [31], and there is a significant difference in the composition of triglycerides between male and female schistosomes, with TG (52:1) being more abundant in females, while TG (58:6) is more abundant in males [32]. In addition, a significant difference was found in the uptake of the fluorescent substance hemoglobin between male and female schistosomes [33].
The application of non-fluorescent substrate chromogenic methods (such as HRP/alkaline phosphatase [AP]-labeled secondary antibodies) for localized signal amplification of schistosome proteins can effectively avoid the interference from AF on localized signals. However, non-fluorescence methods will also suffer from interference by the red blood cell products in the worm gut [34]. In a previous study, the localized signal of schistosome proteins was amplified using fluorophore-labeled secondary antibody in IFA. However, due to the lack of an effective AF reduction method used in the assay, a diffuse background signal was present and interfered with positive fluorescence signal recognition [35]. To increase our understanding of the phenomenon and characteristics of schistosome AF, some researchers have labeled the AF and immunofluorescence signals in schistosome IFA based on previous experience [25]. However, experience sometimes limits the accuracy of true positive signal detection, especially when the immunofluorescence signal overlaps with the AF signal. In our study, through reagent screening and condition optimization, a suitable method for AF reduction in male and female schistosomes was developed. Combined with the IFA developed previously [15], we established an effective AF reduction-based IFA which significantly reduced the AF background signal (Additional file 3: Figure S3, Fig. 4a and b EGFP column) and highlighted the immunofluorescence signal (Fig. 4).
The accuracy of immunofluorescence localization of schistosome proteins relies on the ability to reduce noise from AF and improve the specificity of the antigen-antibody reaction. In this study, the use of reagent screening and condition optimization (Figs. 2, 3, and Additional file 1: Figure S1) enabled us to develop a suitable method for AF reduction in both male and female schistosomes. Applied in IFA, AF was effectively reduced ( Fig. 4 and Additional file 3: Figure S3). Of course, our attention should also be focused on the specificity of antibodies when immunofluorescence localization is performed on one specific protein. In this study, the specific reaction of anti-SjCRT antibody was verified using the WB method (Additional file 2: Figure S2). The results showed the presence of a single band, the weight mass conformed to the theoretical calculated value, and it was consistent with a previous study [36]. SjCRT is one of the immunostimulatory molecules found to induce a T helper type 1 (Th1)-polarized immune response in mice [36], and the peptide sequences of SjCRT were detected in excretory/secretory products of S. japonicum by proteomics [37]. Therefore, S. japonicum in the vein of mice could regulate the polarization of Th1 cells by excreting SjCRT. The main excretory organs of schistosomes are the tegument and anterior esophageal region [38,39], and previous omics studies have shown that SjCRT is expressed in the tegument [40]. In this study, SjCRT was observed in the tegument of male and female worms as well as in the anterior end of the esophagus of female worms by IFA (Fig. 4). Our results further confirm that SjCRT is a protein produced by the excretory organs of S. japonicum.
In this study, only mature S. japonicum male and female worms were used. However, AF has been detected in different kinds of flukes. For example, AF was seen in the vitelline glands [14,41] and eggs [42,43] of S. mansoni and Schistosoma haematobium, in the eggs of Clonorchis sinensis [44,45] and Opisthorchis viverrini [46], and in the vitelline glands of Fasciola gigantica [47,48]. In one study, to minimize AF, 15-dayold C. sinensis flukes exhibiting no visible background fluorescence were used for the IFA [49]. However, for samples with AF, increasing the signal-to-noise ratio enables accurate localization of the target molecules. Our method, by reducing AF noise, facilitates improved signal-to-noise ratio and can perhaps be applied to IFA in other flukes.
In summary, both female and male schistosomes have autofluorescence (AF), but the methods for effective AF reduction are different. Our results suggest that 50 mM CuSO 4 reduces AF in females and 0.5% SBB reduces AF in males. The application of this method in IFA of schistosomes can obviously reduce AF and highlight the IF signal. Therefore, our method can improve the accuracy of functional localization for schistosome proteins and provides a foundation for IFA in other flukes.