Journal of Fluorescence

https://doi.org/10.1007/s10895-018-2217-4

ORIGINAL ARTICLE

Spectral Characteristics of Autofluorescence in Renal Tissue

and Methods for Reducing Fluorescence Background in Confocal Laser
Scanning Microscopy
Yang Zhang1 · Yang Wang1,2 · Wei‑Wei Cao2 · Ke‑Tao Ma1,2 · Wei Ji1 · Zi‑Wei Han1 · Jun‑Qiang Si1,2 · Li Li1,2

Received: 28 November 2017 / Accepted: 16 February 2018

© Springer Science+Business Media, LLC, part of Springer Nature 2018

Abstract
Significant autofluorescence (AF) of renal tissue is one of the major causes restricting the use of immunofluorescent staining.
This study aimed at controlling renal tissue AF and testing an effective method for optimizing specific signals. In the present
study, we observed emergence of strong AF in all renal cells under different fluorescent channels. Significant concentration-
dependent reduction in AF of kidney tissue was observed with the use of sodium borohydride ­(NaBH4) and Sudan black B
(SBB) alone (p < 0.05). Under maximum effective concentration, semi-quantitative analysis revealed that inhibitory effect of
SBB on AF was superior to that of ­NaBH4 (P < 0.01). When the two chemicals were combined, we observed that background
can be reduced, and specific staining can be optimized at optimum concentration. Intensity of renal tissue was examined by
confocal λ scanning, which showed that peaks were located at the range of approximately 480 − 590 nm and similar to those
of flavin and lipofuscin. These results indicated that combined use of ­NaBH4 and SBB, when targeted at different sources
of AF in renal tissue, is the most effective means of reducing background and preserving specificity of fluorescent labels. In
addition, this method does not interfere with various steps of immunofluorescence experiments.

Keywords  Autofluorescence of renal tissue · Immunofluorescence · Sudan black B · Sodium borohydride · Confocal λ
(lambda) scanning

Introduction increasing attention, researchers have observed that strong

Different from fluorescent signals obtained by adding exog-
enous markers, autofluorescence (AF) refers to the natural
fluorescence produced by cells, tissues and organisms when
exposed to ultraviolet radiation and is an intrinsic character-
istic of tissues and cells [1]. In recent years, as kidney AF
and application of laser confocal microscopy have gained
Yang Zhang and Yang Wang contributed equally to this work.
* Jun‑Qiang Si
sijunqiang@shzu.edu.cn
* Li Li
lily7588@163.com
1
Department of Physiology, Shihezi University Medical
College, 192 Beier Road, Shihezi, Xinjiang 832002,
People’s Republic of China
2 reasons, different chemical treatments are also employed.
The Key Laboratory of Xinjiang Endemic and Ethnic
Diseases, Medical College of Shihezi University,
Shihezi 832002, China
fluorescence background in kidneys represents a well-recog-
nized difficulty in immunofluorescence assays. Monitoring
intensity and spectrum of AF is an effective way of assessing
severity of renal tubular necrosis and detecting the degree
of damage in ischemic kidneys [2–4]. The high background
caused by combination of AF and nonspecific antibodies
acts as barrier to immunofluorescence analysis, concealing
or interfering with specific fluorescent signals and subse-
quently resulting in false positive results [5–7].
Generation of renal AF results from the interaction
between endogenous fluorophores and exogenous fac-
tors caused by tissue-processing techniques [8]. Several
chemical treatments have been recently shown to reduce
intrinsic fluorescence of brain tissue [9], pancreatic tissue
[10], and enamel [7]. Intensity of tissue fluorescence is
influenced by physiological, morphological, and biochemi-
cal changes. As various tissues produce AF for different
To our knowledge, few studies on this subject are avail-
able; we applied confocal λ (lambda) scanning technology

13
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to analyze spectral features of renal AF and used two kinds
of chemicals, namely, ­NaBH4 and SBB, to tackle kidney
sections. By performing this method, we observed AF
features and determined methods for minimizing AF and
optimizing specific labels to lay the foundation for applica-
tion of immunofluorescence in renal tissue.
Materials and Methods
The material method is generally divided into the fol-
lowing four parts: (1) preparation of animal models and
tissue sections; (2) preparation of reagents for immu-
nofluorescence and de-autofluorescence chemicals; (3)
immunofluorescent antibody staining; and (4) imaging
and data analysis. Detailed procedures and precautions
are described below.
Animals and Tissue Sections
Female Sprague–Dawley rats weighing 220 − 300  g,
were purchased from Xinjiang University Center for
Animal Experiment, Xinjiang, China, for this study.
All experimental protocols conformed to both interna-
tional and Shihezi Medical College guidelines on ethical
use of animals, and all efforts were exerted to mini-
mize the number of animals used and their suffering.
Experimental groups were divided into control and acute
ischemia–reperfusion injury (IRI) groups (n = 10 for
each group). Renal ischemia was induced by unilateral
clamping of the renal pedicle with a microvascular clip
for 45 min, and circulation was subsequently restored by
removing clips as previously described [11, 12]. After
reperfusion for 6 h, normal and ischemic kidney tissues
were removed. Some tissues were stored in Tissue-Tek
and cut into 4 µm paraffin sections. Other tissues were
fixed in 10% neutral buffered formalin (G2161; Solar-
bio; Beijing; China) and then embedded in paraffin for
storage.
Immunofluorescence
Preparation of Slides Before Immunofluorescent Staining
To remove paraffin from tissue sections, the slides
were immersed in xylene twice for 5 min followed by
decreasing concentrations of ethanol (5 min × 1 in 100%,
5 min × 1 in 90%, 5 min × 1 in 80%, and 5 min × 1 in 70%
ethanol), and then fully hydrated in distilled water.
13
Journal of Fluorescence
Antigen Retrieval for Increasing Incidence
of Immunofluorescence Positive Consequence
Heat and high pressure that induce epitope retrieval can
reveal antigen in formalin-fixed and paraffin-embedded kid-
ney tissues [13]. Citrate buffer (0.01 mmol/L, pH 6.0) was
boiled for 3 min in a microwave at high temperature and
placed in a preheated autoclave. Sections were immersed
in citrate buffer and placed in an autoclave at high tempera-
ture and pressure for 8 min, after that naturally cooled to
room temperature. Subsequently, 3% hydrogen peroxide
was added and incubated for 20 min at room temperature.
Finally, sections were soaked in phosphate-buffered saline
(PBS) (5 min × 3).
Immunofluorescent Staining
After pretreatment in 10% normal fetal calf serum at 37 °C
for 1.5 h, sections were incubated with primary anti-CD4
rat monoclonal antibodies (1:50; ab25475; Abcam; Cam-
bridge, UK) in a humid environment at 4 °C overnight.
The sections were rinsed with PBS, incubated with 10%
normal fetal calf serum at 37 °C for 1.5 h, and incubated
in goat anti-mice IgG conjugated with fluorescein isothio-
cyanate (FITC) (1:400; ab6840; Abcam; Cambridge, UK)
at 37 °C for 1 h. Subsequently, the sections were washed
with PBS buffer with Tween-20 (P1379; Sigma–Aldrich,
Merck KGaA; USA) and incubated in propidium iodide (PI)
(P4170; Sigma–Aldrich, Merck KGaA; USA) for 5 min to
stain the nuclei. All antibodies were diluted in 10% normal
fetal calf serum as blocking reagent. Sections were examined
under a laser confocal microscope (LSM 710, Carl Zeiss
Microscope, Jena, Germany).
Reagents for Diminishing AF
Sodium Borohydride ­(NaBH4)
Tissue sections were treated with ­NaBH4 (0%, 0.1%, 0.5%,
1%, 5%, 10% in PBS; 71320; Sigma–Aldrich, Merck KGaA;
USA) thrice for 10 min after incubating tissues in 3% hydro-
gen peroxide but before incubating them with primary anti-
bodies. Importantly, the chemicals were set up on ice when
used.
Sudan Black B (SBB)
Tissue slides were immersed in different concentrations of
SBB solutions (0%, 0.1%, 0.2%, 0.4%, and 0.5%; 199664;
Sigma–Aldrich, Merck KGaA; USA), which were prepared
with 70% ethanol for 20 min. The SBB solution concealed
AF after incubation with secondary antibody markers. To
Journal of Fluorescence
Fig. 1  AF in renal tissues sections examined by using a CLSM.
Images were acquired at excitations of 488 and 543 nm in FITC and
TRITC channels with confocal laser scanning microscopy. Differen-
tial interference contrast (DIC) images were acquired by excitation at
473 nm. a − c1, confocal images of AF in renal cortical areas. a2 − c2,
prevent excessive staining of SBB, the slides were subse-
quently rinsed with 50% ethanol.
Imaging and Data Analysis
Image Analysis
The sections were examined with an Olympus IX71 micro-
scope (Olympus, Hamburg, German), which was equipped
with four kinds of excitation laser, namely, yellow, cyan,
green, and red excitation lasers at 514, 458, 488, and
543 nm, respectively. All images were acquired with a Zeiss
confocal images of AF in the section of renal medulla. Red arrows
represent blood vessel, yellow arrows represent glomerulus, and
blue arrows represent renal tubules. Wavelength of excitation: FITC,
488 nm; TRITC, 543 nm. Scale bar: a − c: 50 µm, a1 − c2: 10 µm
LSM Image Examiner (Olympus, Hamburg, German) and
Adobe Photoshop 7.0 (Adobe Systems, San Jose, California,
USA).
Statistical Analysis
Data were analyzed by SPSS version 17.0 for Win-
dows (SPSS Inc, Chicago, IL, USA) and expressed as
mean ± standard deviation. For parametric data, compari-
son of different groups was performed by one-way ANOVA,
followed by Tukey’s post hoc test for multiple comparisons.
Student’s t-test was used for two-mean comparisons. Date
13

Fig. 2  Bar graphs showing average fluorescent intensity in FITC
and TRITC channels under the same imaging conditions. Data are
expressed as mean ± standard deviation (SD). N = 6. *p < 0.05 versus
FITC channel
were visualized using GraphPad Prism 5 (GraphPad Soft-
ware Inc, San Diego, CA, USA). Differences in median data
values with p-values < 0.05 were considered significant.
Results
Characteristics of AF Background in Renal Tissue
As shown in Fig. 1, AF was observed in all renal cells of
the normal kidney under FITC and tetramethylrhodamin
isothiocyanate (TRITC) channels. Intense AF was emitted
in cortexes (Fig. 1, a − c1), medullas (Fig. 1, a2 − c2), and
blood vessels (red arrows in Fig. 1) of the kidney when visu-
alized under confocal laser scanning microscopy (CLSM).
AF was evidently brighter in the FITC channel compared
with that in the TRITC channel under the same imaging
conditions (Fig. 2).
Methods for Controlling AF in Renal Tissues
To determine whether renal tissue also emitted AF in other
fluorescence channels, the slides were examined for yellow
(YFP) and cyan-blue fluorescent protein (CFP) channels.
We noted that the sections untreated with chemicals all dis-
played AF in the following FITC and TRITC fluorescent
protein channel (Fig. 3, a − e).
Effect of ­NaBH4 on AF
Serial sections of paraffin-embedded normal kidney tissues
were prepared. These sections were treated with increasing
13
Journal of Fluorescence
concentrations of ­NaBH4 to detect changes in fluorescence
intensity at various concentrations of ­NaBH 4. Results
revealed that treatment with ­NaBH4 slightly reduced AF
regardless of excitation wavelength (Fig. 3). Average fluores-
cence intensity of treated samples demonstrated a significant
difference compared with slices without chemical treatment.
A decreasing trend in AF intensity was observed as concen-
tration of chemicals increased (Fig. 4). Additionally, when
concentration of ­NaBH4 reached 10%, tissues sections were
easily removed (data not shown).
Effect of SBB on AF
As treatment of ­NaBH4 slightly reduced AF of tissues as pre-
viously mentioned, we also used SBB, which is commonly
used to conceal fluorescence, to investigate its role in AF of
kidney tissues. SBB staining after antibody incubation and
prior to cover slip mounting markedly reduced the AF gener-
ated by these natural sources (Fig. 5). Average intensity of
AF was effectively reduced in renal tissues excited at 514,
458, 488 and 543 nm in an SBB dose-dependent fashion.
Renal tissue AF in the TRITC channel was not observed
when tissues were treated with 0.5% SBB. Intensity of tissue
staining by SBB was visualized using bright-field micros-
copy. Afterward, optimized SBB staining procedure was
used for detailed quantitative analysis of AF-quenching
effect (Fig. 6).
Comparison of the Effects of ­NaBH4 and SBB on AF
Under maximum effective concentration of ­NaBH4 and SBB,
fluorescence semi-quantitative results (Fig. 7) revealed the
more significant effect of SBB on AF than ­NaBH4.
Immunofluorescence Staining After ­NaBH4 and SBB
Combination
Based on the above results, we selected the two chemicals
with the best concentration, namely, ­NaBH4 and SBB, to
process sections from the rat IRI model, these samples
were stained afterward. Results indicated that the specific
immunofluorescence was impossible to distinguish from
background AF through anti-CD4 antibody labeled with
FITC lacking 10% ­NaBH4 and 2% SBB (Fig. 8, a1 − c1).
However, improved images (Fig. 8, a2 − c2) were obtained
after employing this method to reduce AF, optimizing anti-
body staining to the utmost extent compared with treated
IRI group (Fig. 8, a1 − c1). Compared with treated control
group (Fig. 8, a − c), the brilliant fluorescence was found in
sections of the IRI rat model after treatment (yellow circles
in Fig. 8, a1 − c2). In summary, treatment with N ­ aBH4 and
SBB yielded the expected effective results for single staining
with anti-CD4 antibody.
Journal of Fluorescence
Fig. 3  Effect of N
­ aBH4 on AF was examined at different concentra-
tions (0%, 0.1%, 0.5%, 1%, 5%, and 10%). Treatment with N ­ aBH4
(a1 − e5) led to a slight reduction in AF background. All images
were obtained under the same magnification. YFP channel: (a1 − a5);
CFP channel: (b1 − b5); FITC channel: (c1 − c5); TRITC channel:
(d1 − d5); DIC channel: (e1 − e5). Wavelength of excitation: YFP,
514  nm; CFP, 458  nm; FITC, 488  nm; TRITC, 543  nm. Controls
(untreated sections, a, b, c, d, and e). Scale bar: 50 µm
13
 Journal of Fluorescence

Fig. 4  Changes in fluorescence
intensities of renal tissues
treated with ­NaBH4 different
concentrations. Fluorescence
intensity profiles of renal tissues
from channel (a) YFP, (b) CFP,
(c) FITC, (d) TRITC, and (e)
all four channels. Statistical
analysis was performed by using
one-way ANOVA followed by
Tukey’s test. Data are expressed
as mean ± SD. N = 6. *p < 0.05
versus untreated group,
#
p < 0.05 versus 0.1% concen-
tration, &p < 0.05 versus 0.5%
concentration, Δp < 0.05 versus
1% concentration, and □p < 0.05
versus 5% concentration

Spectral Analysis of AF Intensity in Normal Kidney
Tissues
AF intensity in renal tissues was measured using a laser
confocal scanning microscope under the following channels.
In CLSM λ scanning mode, six points in the image were
randomly selected and plotted based on their AF values.
Dominant peak of AF appeared at about 540 nm, when kid-
ney tissue AF was examined through a yellow channel with a
wavelength of 514 nm (Fig. 9a). Similarly, four AF peaks in
the range of 478–649 nm emission wavelength was observed
when renal sections delivered cyan fluorescence at 458 nm
excitation. We also observed that most peaks appeared at
505, 530, 555, and 580 nm (Fig. 9b). As shown in Fig. 9c,
when excited at 488 nm, we found that the main peak of
autofluorescence may appear before 500  nm. Similarly,
an AF peak at the range of 560 − 700 nm excitation light
emerged when renal sections emitted red fluorescence of
543 nm excitation (Fig. 9d). In summary, the peak value
was similar to AF peaks of flavin and lipofuscin, which were
suggested to be the substances that triggered intrinsic fluo-
rescence in renal sections.
Discussion
Appearance of AF in biological tissue cells poses a remark-
able challenge in immunofluorescence assays. Therefore,
exploring the source of AF in renal tissues is particularly
important. In our initial investigation, results indicated that
strong fluorescence mainly occurs in the glomeruli, renal
vessels, especially in renal tubules (Fig. 1), coinciding with

13
Journal of Fluorescence
Fig. 5  Concentration-dependent reduction of AF by SBB. Intensity of
AF of these SBB treated sections was evaluated with the four fluo-
rescence channels. AF intensity of the four fluorescent channels of
rats decreased after administration of diverse concentrations of SBB
compared with the normal group. (Low SBB concentrations (< 0.1%)
provided insufficient reduction of autofluorescent pigments. However,
high SBB concentrations (> 0.2%) adequately reduced autofluores-
previous studies [8, 14]. Baschong et al. have demonstrated
that red blood cells (RBCs) can also contribute significantly
to AF [5]. In this study, punctate fluorescence in glomeruli
and interstitium was assumed to be derived from RBCs (yel-
low arrows in Fig. 1) [8, 15]. A few studies have proven that
medullary AF significantly increases after IRI or renal tumor
cent pigments in sections). All images were obtained under the same
magnification. YFP channel: (a1 − a4); CFP channel: (b1 − b4); FITC
channel: (c1 − c4); TRITC channel: (d1 − d4); DIC channel: (e1 − e4).
Wavelength of excitation: YFP, 514  nm; CFP, 458  nm; FITC,
488 nm; TRITC, 543 nm. Controls (untreated sections, a, b, c, d, and
e). Scale bar: 50 µm
and can be used as an indicator of the degree of disease dam-
age [3, 16–18]. Interestingly, as shown in Fig. 8, a1 − c2, we
detected some irregular and extremely bright substances in
medullary AF of the IRI model when compared with the
control group. These strong fluorescent substances, which
scattered in the tubulointerstitium, were considered injured
13
 Journal of Fluorescence

Fig. 6  Changes in fluorescence
intensities of renal tissues
treated with SSB at different
concentrations. Fluorescence
intensity profiles of renal tissues
from channel (a) YFP, (b) CFP,
(c) FITC, (d) TRITC, and (e)
all four channels. Statistical
analysis was performed by using
one-way ANOVA followed by
Tukey’s test. Data are expressed
as mean ± SD. N = 6. *p < 0.05
versus untreated group,
#
p < 0.05 versus 0.1% concen-
tration, &p < 0.05 versus 0.2%
concentration, and Δp < 0.05
versus 0.4% concentration

interstitial cells, protein casts [8], and infiltrating inflamma-

Fig. 7  Semi-quantitative analysis showed the effect of ­NaBH4 and
SBB at maximum concentrations on kidney AF under different fluo-
rescent channels. Data are expressed as mean ± SD, n = 6. *p < 0.01
versus ­NaBH4
tory cells. Some studies have also revealed that AF occurs
in structural proteins, especially in collagen and elastin [19,
20], which are the major luminescent groups in the extracel-
lular matrix [21]. Thus, we have identified that collagen and
elastin [1] are sources of renal vascular AF, as reported in
other tissues [14].
The source of AF in kidney tissue is very complex.
Cell AF mainly depends on intrinsic fluorophores, such as
lipofuscin and emodin. Lipofuscin is widely distributed in
various tissues and body organs, such as sweat glands [22],
brain [23, 24], heart [25], liver [26], uterus, intestine, and
kidney [27, 28]. Recently, a number of research studies have
shown that accumulation of lipofuscin is closely related to
cell senescence. Lipofuscin is a pigmented and spontane-
ously occurring fluorophore produced by lipid oxidation

13
Journal of Fluorescence
Fig. 8  Single-labeling immunofluorescence of CD4 (green, a − a2) in
diseased kidney after eliminating AF background. PI was applied to
stain nuclei (red, b − b2). Merged images (c − c2) were obtained with
or without treatment of quenching AF. Yellow arrows and yellow
circles represent specific immunofluorescence signals and brilliant
and features an excitation peak at 340 − 395 nm, resulting
in natural fluorescence peaks close to 450 and 600 nm [1,
29, 30]. The presence of flavonoids in most metabolic path-
ways is essential in metabolic pathways of sugar, protein, fat,
and nucleic acids [31]. Inherent fluorescence of flavonoids is
derived from oxidation reaction, whereas peak of excitation
light appears at 360 and 450 nm, with an AF wavelength at
520 nm [31, 32]. In CLSM scans, peaks of kidney section
AF were close to 480 − 590 nm. These results are similar
to the peaks of flavin and lipofuscin; thus, we speculate
that flavin and lipofuscin may be the main sources of AF in
rat kidney sections [21, 25, 27]. AF is generated either by
fluorescent substance respectively in FITC channel. Wavelength of
excitation: FITC, 488 nm; TRITC, 543 nm. Treated Control (sections
in normal renal tissue): a − c; Untreated IRI (sections in IRI renal tis-
sue): a1 − c1; Treated IRI: a2 − c2. IRI, ischemia reperfusion injury.
Scale bar: a − c2: 10 µm
endogenous groups or by histological processing. Previous
studies showed that tissue fixative and paraffin embedding
agents contribute to fluorescence, due to the reaction of alde-
hyde groups with amino acids or proteins in tissues [33, 34].
Regarding the fact that tissue fixatives and paraffin embed-
ding agents produce AF, our experiment did not establish an
additional control group to prove separately.
Currently, several strategies for controlling AF have been
put forward [5, 9]. We divided the protocols for elimina-
tion of AF into three categories namely, chemically reduc-
ing compounds [33, 34], dyes with affinity to stain vari-
ous tissue components [9], and photobleaching [35]. As
13

Fig. 9  Spectrum analysis of AF of normal rat kidneys using confo-
cal microscope λ scanning. Six points were selected randomly to plot
curves of AF using LSM software under (a): YFP; 514 nm, (b): CFP;
previously illustrated in other histological sections, ­NaBH4
has long been applied to correct unnecessary fluorescence in
formalin-fixed or glutaraldehyde-fixed tissues [33, 34]. The
exact mechanism of N ­ aBH4 transpires via the interaction
between aldehydes and ketones, causing their reduction into
their respective alcohols. However, the usage of ­NaBH4 in
other tissue specimens was restricted due to its adverse effect
on mounting quality of treated tissues. In the present study,
we tested treatment of kidney tissue at different ­NaBH4
concentrations and observed that as ­NaBH4 concentration
increased, AF intensity decreased. Although to a certain
extent, as the concentration of N
­ aBH4 increases, AF in renal
tissue presents a downward trend, it also has a detrimental
effect. When N­ aBH4 concentration exceeded 10%, the tissue
was easily removed from the slide, or AF intensified due to
induction of brighter fluorescence, such as in erythrocytes
[36], suggesting that this method is inappropriate for renal
tissues. A potential explanation of the low effectiveness of
13
Journal of Fluorescence
458 nm, (c): FITC; 488 and (d): TRITC; 543 nm excitation waves
(one point represents to one pixel value)
­NaBH4 may lie in the fact that AF in kidney tissues mainly
results from the actions of endogenous substance and not
just by tissue fixation.
Unlike ­NaBH4, SBB is a conventional, fat-soluble four-
nitrogen dye that can mask AF of tissues. Based on its ability
to dissolve the main AF-inducing lipid components, such as
lipofuscin, SBB is often used to eliminate AF in the brain
or myocardium [37, 38]. Remarkably, SBB possesses strong
light absorption property in the visible range and features no
AF at 640 nm [24, 39]. In this study, SBB staining was not
used to directly describe the observed signals but to mask
unnecessary signals and to improve signal-to-noise ratio of
samples. Schnell et al. [25] showed concentration-depend-
ent reduction in lipofuscin-like AF by using SBB, showing
agreement with our results. They also revealed that low SBB
concentrations (< 0.01%) resulted in insufficient reduction
in autofluorescent pigments, whereas high concentrations
(> 1%) caused adequate reduction in lipofuscin-like AF in
Journal of Fluorescence
neural tissues [5]. Similarly, we discovered that an SBB
concentration of 0.5% can almost completely conceal auto-
fluorescent pigments in renal tissues and also mask specific
signals. However, this phenomenon can be reversed by treat-
ment with a more soluble reagent, which can wash off the
SBB stain. In summary, these data showed that at a certain
range, higher SBB concentrations not only cause better fluo-
rescence suppression on the background, but also increase
the degree of attenuation of signals, indicating that optimum
concentration of 0.5% is inadequate for immunofluorescence
staining. Although SBB treatment provided more effective
results than using N­ aBH4 alone, high SBB concentration
affected observation of specific signals.
Additionally, other treatments, including photobleaching
(i.e., exposure to ultraviolet radiation) and glycine, were
applied to detect their effect in reducing kidney AF. How-
ever, results revealed low effects (data not shown).
All these techniques come with their own drawbacks.
Application of chemicals also reduces intensity of immu-
nofluorescent labeling but requires a balance between AF
reduction and antigen visualization. To optimize immu-
nostaining of renal tissues, we combined the two meth-
ods mentioned above to process sample slices. We further
observed that when combined concentration (10% ­NaBH4
and 2% SBB) was appropriate, magnitude of background
reduction was sufficient in several cases to detect specific
signals by direct immunofluorescence. Though some bright
AF was still present. The potential factors that contribute
to bright AF should be noted; however, scholars currently
present differing opinions. Some believe that this phenom-
enon due to the use of ­NaBH4 causing brighter red cell AF
[36], whereas others indicate that bright substances should
be attributed to protein casts [8]. More studies should be
conducted to understand the mechanisms behind AF.
In conclusion, we conducted a number of experiments to
optimize immunofluorescence procedure and minimize the
AF generated from renal tissues. Currently, to our knowl-
edge, commonly used fluorescence secondary antibodies,
include FITC and TRITC, in which isothiocyanates play an
important role in covalent linking with antibodies and amino
groups. No study reported that these methods involved in
attenuating AF will affect covalent binding between antibod-
ies, suggesting feasibility of these histochemical methods
for blocking AF. Notably, SBB solution preparation, dyeing
temperature, dyeing time, the number of rinses, and incu-
bation period can affect intensity of fluorescent protein or
fluorescent dyes. Most importantly, to ensure the normal
nature of fluorescent proteins or fluorescent dyes, avoiding
the use of substances with strong redox properties is nec-
essary because they undergo a special redox reaction with
fluorescent proteins or fluorescent dyes. Finally, to main-
tain fluorescence intensity of fluorescent proteins or dyes,
sections should be processed as fast as possible at the lowest
possible temperature.
Acknowledgements  The present study was supported by National Nat-
ural Science Foundation of China (No. 81560175 and No. 81260159),
the High Level Talent Research Project of Shihezi University (grant no.
RCSX201705), and Xinjiang Autonomous Region Graduate Research
and Innovation Project (grant no. XJGRI2017033).
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