Quenching autofluorescence in tissue immunofluorescence

Background: Immunofluorescence (IF) is one of the most important techniques where fluorochromes conjugated to antibodies are used to detect specific proteins or antigens. In tissue sections, autofluorescence (AF) can lead to poor quality images that impair assessment. The placenta is a pivotal extra-embryonic organ in embryo development, where trophoblasts make up a large proportion of the cells. Teratoma formation is one of the critical assays for pluripotent stem cells. Methods: We tested whether ultraviolet (UV), ammonia (NH3), copper (II) sulfate (CuSO4), Trypan Blue (TB), Sudan Black B (SB), TrueBlackTM Lipofusin Autofluorescence Quencher (TLAQ) and combinations of these treatments could reduce AF in paraffin and frozen sections of placenta and teratoma in FITC, Texas Red and Cy5.5 channels. Results: We found that UV, NH3, TB and CuSO4 quenched AF to some extent in different tissue and filters, but increased AF in Texas Red or Cy5.5 channels in some cases. SB and TLQA exhibited the most consistent effects on decreasing AF, though TLQA reduced the overall IF signal in placenta sections. Not all combined treatments further reduced AF in both placenta and teratoma sections. Conclusions: SB and TLAQ can effectively quench AF in placenta and teratoma IF. Jian Yang ( ), Pentao Liu ( ) Corresponding authors: jy4@sanger.ac.uk pl2@sanger.ac.uk : Conceptualization, Data Curation, Investigation, Methodology, Writing – Original Draft Preparation, Writing – Review & Author roles: Yang J Editing;  : Data Curation, Methodology, Writing – Review & Editing; : Data Curation, Writing – Review & Editing; : Yang F Campos LS Mansfield W Methodology; : Methodology; : Methodology, Writing – Review & Editing;  : Funding Acquisition, Supervision, Writing – Skelton H Hooks Y Liu P Review & Editing Competing interests: No competing interests were disclosed. Yang J, Yang F, Campos LS How to cite this article: et al. Quenching autofluorescence in tissue immunofluorescence [version 1; Wellcome Open Research 2017,  :79 (doi: ) referees: 1 approved with reservations] 2 10.12688/wellcomeopenres.12251.1 © 2017 Yang J  . This is an open access article distributed under the terms of the , which Copyright: et al Creative Commons Attribution Licence permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. This work was supported by the Wellcome Trust [206194]. Grant information: The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. 04 Sep 2017,  :79 (doi: ) First published: 2 10.12688/wellcomeopenres.12251.1 1 1 1 2 2 1

Introduction Immunofluorescence (IF) is an important assay used in stem cell research.By incubating cells or tissues with fluorescenceconjugated antibodies, the identity of cells and their descendants to tissues and organs can be identified and quantified by fluorescence microscope or flow cytometry (Manos et al., 2011;Stack et al., 2014).Though this method is widely used and well developed, so far, autofluorescence (AF) can present a serious issue to obtain clear and conclusive data for some tissue samples (Abuduwali et al., 2013;Yang & Honaramooz, 2012).AF is the natural and fixative induced fluorescence occurring in cells and tissues with a wide spectrum between 450nm and 650nm, which overlaps with the commonly used fluorophores in IF (Monici, 2005;Viegas et al., 2007).Many factors are known to contribute to AF, such as sample fixation, tissue components, lipofusin, elastin and collagen (Banerjee et al., 1999;Del Castillo et al., 1989;Monici, 2005).Various protocols have been developed to tackle this issue in order to improve imaging quality (Baschong et al., 2001;Davis et al., 2014;Viegas et al., 2007).
The placenta is a vital extra-embryonic organ that serves as the interface between foetal and maternal environments to exchange gas, nutrients and carry away wastes.It also secrets pregnancy related hormones and growth factors, and provides immuneprotection for the foetus.The placenta has various types of cells, including trophoblasts, vascular endothelial cells and blood cells; among which, trophoblasts are the structural and functional components of the placenta, and are the most important cell type.
Trophoblasts are the progeny of cells in the trophectoderm.Defects in foetal, maternal components and trophoblasts in the placenta affect its functions and can cause severe pregnancy complications (Maltepe & Fisher, 2015;Rossant & Cross, 2001).
One functional assay used to demonstrate the differentiation potential of human pluripotent stem cells is teratoma formation.When pluripotent stem cells are implanted at ectopic sites in immunocompromised mice, for example skin or kidney capsule, the cells differentiate into various cell types (Cunningham et al., 2012;Lensch et al., 2007;Muller et al., 2010).
Both placenta and teratoma are important tissues for stem cell and developmental biology.In this study, we employed both physical and chemical approaches to test their effects on quenching AF in placenta and teratoma IF assays.Teratoma formation NSG mice were anesthetised by inhalation with Isoflurane, shaved and cleaned.Analgesia (Carprieve) 0.1ml was administrated by sub-cutaneous injection.A small incision was made on the left flank of the mouse and the kidney was exposed.An injection needle was used to gently pinch the capsule membrane.1×10 5 H1 hESCs were transferred into the kidney capsule using a fine glass pipette by mouth pipetting.The kidney was placed back into the cavity and the peritoneum and muscular wall was sutured and the skin stapled.Approximately 8 weeks later, a teratoma grew at the injection site.When the teratoma reached 1.2 mm 2 , the mice were euthanized; kidney and teratomas were dissected, fixed overnight in 10% neutral buffered formalin and embedded in paraffin before sectioning.

Animal ethics statement
The same procedure was applied for the sub-cutaneous teratoma formation assay.5×10 6 H1 hESCs were injected subcutaneously into both dorsal flanks of NSG mice.When the size of the teratomas reached 1.2 mm 2 , the mice were euthanized and the teratomas were dissected, fixed overnight in 4% PFA and embedded in O.C.T compound before frozen sectioning.

Immunofluorescence
For paraffin-embedded sections, antigens were retrieved in sodium citrate buffer (10 mM sodium citrate, 0.05% Tween-20, pH 6.0) by microwave for 15 minutes, followed by rinsing with tap water.The slides (both paraffin embedded sections and frozen sections) were then washed in PBS containing 0.25% Triton X-100 (PBST) twice for 5 minutes each, blocked in PBST/1% BSA/5% donkey serum for 1 hour at room temperature, briefly rinsed with PBST and incubated with primary antibody at 4°C overnight.The slides were washed with PBST 3 times for 5 minutes each, blocked in PBST/1% BSA/5% donkey serum for at least 15 minutes, and incubated with AlexaFluor® conjugated secondary antibody for 1 hour at room temperature, then washed with PBST 3 times for 5 minutes each.The sections were mounted in a small drop of Prolong Gold® antifade mountant with DAPI, covered with a coverslip and sealed with nail varnish.

Treatments. Ultraviolet (UV):
Sections were irradiated with UV, 2 × 15W, 312nm, on ice for 2 hours.Paraffin wax was then removed by placing in xylene and rehydrated for immunofluorescence.

Sudan Black B (SB):
After blocking with serum and BSA, sections were immersed in 0.1% SB for 20 minutes at room temperature.
Sections were then washed with PBST 3 times, for 15 minutes each; jet wash was carried out before antibody incubation.
Trypan Blue (TB): After blocking, sections were treated with 0.05% TB for 15 minutes at room temperature, washed with PBST 3 times, for 15 minutes each, and then incubated with antibody.

TrueBlack lipofusin autofluorescence quencher (TLAQ):
After blocking, sections were treated with TLAQ for 30 seconds at room temperature, then washed in PBS 3 times, for 15 minutes each.The sections underwent all the following steps for IF using PBS only for antibody incubation and wash.
Copper (II) Sulfate ( CuSO4): Before or after IF, sections were treated with CuSO4 5 mM in 50 mM ammonia acetate for 90 minutes, then washed with PBST 3 times, for 15 minutes each, then mounted for imaging and analysis.
Combined treatment: Different combinations of chemical treatment are listed in Table 1.
Digital imaging evaluation.The sections were examined under a Leica DM5000B microscope equipped with narrow bandpass filters for FITC (488nm), Texas Red (594nm), Cy5.5 (630nm) and DAPI (405nm) fluorescence.Images were captured via a monochrome digital camera (ORCA-03G, Hamamatsu) and processed with the SmartCapture software (Digital Scientific UK).
The same settings for acquisition mode and exposure time were applied to all samples that had undergone different treatments.

Results
Paraffin sections of 14.5 d.p.c placenta Mouse 14.5 d.p.c placenta sections were treated with UV, NH3, SB, TLAQ or NH3/SB.The sections were incubated with the primary antibody against a trophoblast specific marker Tfap2c 594 nm (Texas Red) and 630 nm (Cy5.5) were evaluated and images were captured with the same setting.The mouse placenta organizes in three layers, i.e labyrinth, junctional zone, and decidua (Favaron et al., 2013).We mainly analysed AF in labyrinth and junctional zone.Results were summarised in Table 2.
UV: Compared with untreated control, UV treatment (in FITC channel) reduced AF in junctional zone, but no visible changes were observed in labyrinth.In Texas Red channel, both junctional zone and labyrinth showed reduced AF.In Cy5.5 channel, there was no difference in junctional zone, but AF was reduced in the labyrinth, although it was enhanced in blood cells (Figure 1A and B).
NH3: In FITC channel, AF was reduced in junctional zone, but no visible difference was seen in the labyrinth.In Texas Red channel, AF was reduced in both junctional zone and labyrinth.In Cy5.5 channel, there was marked reduction in junctional zone AF; the same occurred in the labyrinth, but here AF was enhanced in blood cells (Figure 1C).

SB:
In FITC channel, AF was reduced in both junctional zone and labyrinth.In Texas Red channel, there was marked reduction in junctional zone AF and in labyrinth there was also a reduction in AF, but strong AF was seen in blood cells.In Cy5.5 channel, there was a reduction in AF in junctional zone, but higher AF was seen in blood cells in labyrinth (Figure 1D).

Sequential treatment with NH3 and SB:
In FITC channel, AF was reduced both in junctional zone and in labyrinth.In Texas Red and Cy5.5 channels, in the junctional zone, AF was similar to SB-treated alone sections; in the labyrinth, AF from blood cells was still visible, but lower than in sections treated with SB alone (Figure 1E).

TLAQ:
In FITC channel, AF was markedly reduced, but the Tfap2c specific signals were also affected in both junctional zone and labyrinth.In Texas Red channel, both junctional zone and labyrinth showed reduced AF.In Cy5.5 channel, there was a reduction in AF in junctional zone and labyrinth, but strong AF was seen in blood cells (Figure 1F).

Paraffin sections of kidney and teratoma
Next, we applied the above mentioned UV, NH3, SB, TLAQ and NH3/SB treatments to teratoma sections of hESCs, together with TB, CuSO4 and their combination with SB.The results were listed in Table 3.As the hESCs were injected into the kidney capsule, AF in the kidney background was analysed as well.Cytokeratin 7 was used to detect epithelia components in teratoma (Chu et al., 2000), and the signal was picked up using an AlexaFluor 488 conjugated secondary antibody.
UV: Unlike the placenta, no visible difference was found in AF between treated and control tissues in both kidney and teratoma, in all three channels (Figures 2A and B, Figures 3A and B).
NH3: Both tissues showed reduced AF in FITC channel, but no reduction in Texas Red channel.In Cy5.5 channel reduced AF was observed only in kidney (Figure 2C, Figure 3C).

SB:
In FITC channel, AF was reduced in both kidney and teratoma.
In Texas Red channel, AF was reduced in kidney; but in teratoma, blood cells and basement membranes showed enhanced AF with decreased background.In Cy5.5 channel, AF was generally reduced in both kidney and teratoma, but increased in blood cells and basement membranes (Figure 2D, Figure 3D).

TB:
In FITC channel, AF was reduced in both kidney and teratoma.
In Texas Red channel, kidney showed reduced AF, but in teratoma AF increased.In Cy5.5 channel, higher AF was observed in both kidney and teratoma (Figure 2E, Figure 3E).
CuSO4: When tissues were treated with CuSO4 after blocking, AF was reduced in both kidney and teratoma in FITC channel; in Texas Red channel, low AF was observed in teratoma, whereas in Cy5.5 channel, kidney showed low AF.When the sections were treated with CuSO4 after IF, AF was reduced in FITC channel in both kidney and teratoma; but no difference was observed in Texas Red and Cy5.5 channels (Figure 2F and G, Figure 3F and G).

TLAQ:
In FITC and Texas Red channels, both kidney and teratoma showed reduced AF.In Cy5.5 channel, AF was reduced only in kidney and no difference was observed in teratoma (Figure 2H, Figure 3H).

Combined treatment. NH3/SB:
In FITC and Texas Red channels, there was reduced AF in kidney and teratoma.In Cy5.5 channel, AF was reduced in teratoma, and in kidney, there was reduced AF except in the basement membranes where AF was increased (Figure 2I, Figure 3I).

TB/SB:
In FITC channel, AF was reduced in both kidney and teratoma.In Texas Red channel, no difference in kidney, but increased AF was detected in teratoma.In Cy5.5 channel, increased AF was observed in both kidney and teratoma (Figure 2J, Figure 3J).
CuSO4/SB: CuSO4 treatment followed by SB reduced AF in kidney and teratoma in FITC and Texas Red channels, but in Cy5.5 channel there was increased AF in kidney (Figure 2K, Figure 3K).
SB/CuSO4: SB treatment followed by CuSO4 reduced AF in kidney and teratoma in FITC and Texas Red channels, no difference was observed in Cy5.5 channel in teratoma, whereas in kidney, AF was increased (Figure 2L, Figure 3L).

SB/IF/CuSO4
: SB treatment during IF, then CuSO4 was treated after IF can markedly reduce AF in FITC channel, with reduction in Texas Red channel as well.However, in Cy5.5 channel, there was increase of AF in kidney basement membranes with no difference in teratoma (Figure 2M, Figure 3M).
SB/TLAQ: Marked AF reduction was observed in both kidney and teratoma in all three channels (Figure 2N, Figure 3N).
SB and TLAQ showed consistent effects on all three tissues, so we tested these two reagents on frozen sections as well.
Frozen sections of 10.5 d.p.c placenta Sections were stained with Tfap2c antibody and signals were detected in FITC.The results were summarized in Table 4.

SB:
Compared with untreated control, AF was reduced in both junctional zone and labyrinth in FITC channel.In Texas Red channel, only labyrinth showed reduced AF.In Cy5.5 channel, general AF reduction was observed with increased AF in blood cells (Figure 4A and B).
TLAQ: AF was markedly reduced in both FITC and Texas Red channels.In Cy5.5 channel, AF was reduced in junctional zone and labyrinth, except in blood cells where AF was increased (Figure 4C).

SB/TLAQ:
In FITC channel, AF was markedly reduced, but was accompanied by reduction in the Tfap2c signal as well.In Texas Red channel, both junctional zone and labyrinth showed reduced AF.In Cy5.5 channel, the junctional zone showed reduced AF, whereas in labyrinth general AF reduction was accompanied by increased AF in blood cells (Figure 4D).Frozen sections of teratoma Teratoma sections were incubated with the Cytokeratin 7 antibody and signals were observed in FITC channel.The results were summarized in Table 5.
SB: AF was reduced in FITC and Texas Red channels; but no difference from untreated control was observed in Cy5.5 channel (Figure 5A and B).TLAQ: Similar to SB-treated ones, reduced AF was observed in FITC and Texas Red channels but not in Cy5.5 channel (Figure 5C).
SB/TLAQ: No further AF reduction was observed compared to SB or TLAQ treatment alone in FITC and Texas Red channels.In Cy5.5 channel, no difference was observed either (Figure 5D).

Discussion
In this technical report, we presented analysis results of performing immunofluorescence to detect proteins in the placenta and teratoma sections.Due to the complexity of tissues and the process of tissue section preparation, AF from cells or basement membranes can affect antibody signals detected by fluorochromes (Abuduwali et al., 2013;Banerjee et al., 1999;Baschong et al., 2001;Davis et al., 2014;Duong & Han, 2013;Viegas et al., 2007).Here, we analysed effects of UV and several chemicals, individually or in combinations, on AF in IF.
UV is a photo-bleaching method, which can change the structure of a fluorophore so that it loses its ability to fluoresce (Billinton & Knight, 2001).Quenching with UV has different effects on tissue AF; some authors reported obvious reduction, while others did not observe noticeable differences (Davis et al., 2014;Viegas et al., 2007).Similarly, in the placenta in the present study, UV treatment reduced AF in Texas Red channel in both junctional zone and labyrinth, but had no effects on kidney and teratoma sections in all three channels.
Previously, NH3, TB and CuSO4 were reported to reduce AF in some tissues (Baschong et al., 2001;Loike & Silverstein, 1983;Srivastava et al., 2011;Zhang et al., 2010).NH3 reacts with free formaldehyde to suppress AF in paraffin sections of formaldehyde-fixed, decalcified bone marrow, and may also dissolve negatively charged lipid derivatives, phenols or polyphenols, and hydrolyse weak esters (Baschong et al., 2001).TB can rapidly diffuse into cells after permeabilization, resulting in uniform distribution in the cytoplasm and nucleus, which results in reduced AF when TB molecules are at a proper distance and orientation to autofluorescence or nonspecifically bound to fluorescent molecules (Srivastava et al., 2011).The mechanism of Cu 2+ in AF quenching is not clear.Since it's an excellent electron scavenger, electrons from excited lipofuscin can be transferred to Cu 2+ , or Cu 2+ and lipofuscin can form nonfluorescent complex (Schnell et al., 1999).All three chemicals showed heterogeneous effects in the present study; in placenta, NH3 reduced AF mainly in junctional zone; in kidney, all three channels showed reduced AF; in teratoma, similar effects were achieved with no visible difference in Cy5.5 channel.In kidney, TB reduced AF in FITC and Texas Red channels, but increased AF in Cy5.5 channel, whereas in teratoma sections, AF increased in Texas Red and Cy5.5 channels, but not in FITC channel.The effect of CuSO4 depends on when it is applied during IF; we found that only CuSO4 treatment after IF can reduce AF on FITC in both kidney and teratoma.
SB has been suggested to be a good AF quencher in several reports, which can reduce AF of lipofuscins, fats, triglycerides and lipoproteins (Davis et al., 2014;Erben et al., 2016;Viegas et al., 2007).
SB absorbs at 580nm, so it may absorb a small amount of AF or directly interact with AF species (Davis et al., 2014).We found SB can reduce AF in all three tissues tested in both paraffin and frozen sections, but we detected some increased AF from blood cells and basement membranes in Texas Red and Cy5.5 channels, which is consistent with other reports showing AF was quenched most in FITC channel; in Texas Red channel, the effect is much less, which may be due to the specific excitation wavelength or interference of the high transmission value of this filter with SB treated surface (Erben et al., 2016;Viegas et al., 2007).TLAQ is a commercialized AF quencher, Chan et al. used it to reduce AF in human eye tissue section IF (Chan et al., 2015).In this study, we showed that TLAQ treatment resulted in a reduction in AF, similar to the level produced by SB treatment in all three tissues, but TLAQ treatment was less labouring than SB.However, in placenta, TLAQ treatment reduced the specific signals from the antibody as well.
We also analysed some combined chemical treatments.NH3/SB combination did not further reduce AF in placenta, but in kidney and teratoma, AF in FITC channel was markedly reduced compared to NH3 and SB alone.SB/IF/CuSO4 also further reduced AF in FITC in both kidney and teratoma sections.In Texas Red channel, AF was reduced in teratomas, but AF increased in kidney in Cy5.5 channel.SB/TLAQ treated sections showed marked reduction in all three tissues in both paraffin and frozen sections, but in placenta frozen sections, in FITC, the antibody signal was reduced too.
In summary, we showed that SB is a common quencher that can be used in various tissues.TLAQ is also a good quencher, but it may also quench the real signal from the antibody.For UV and other chemical methods, AF in tissues can be reduced to some extent.However, when choosing the chemicals for AF reduction, the property of the tissues, an adequate fluorochrome, and the channel of acquisition to use all need be considered in order to accurately detect antibody signals.Combinations of chemicals do not always further reduce AF. 1.

Open Peer Review
Current Referee Status: The reviewers congratulate the authors for performing this research study and presenting the data as a scientific paper.
The reviewers present few serious concerns regarding this paper; The paper lacks a new and innovative idea; however, the studied methodologies and research findings support to advancement in scientific research field.
The paper is completely based to develop methodology.Hence, it is considered as a "Method Articles" or "Technical Reports", not as a "Research Articles".
The critical issue is that nowhere in the manuscript the authors have discussed how they are going to apply these research findings in human clinical pathology.
This research needs to apply human tissue samples to extrapolate the research findings application in clinical use.
It needs to be clarified the findings application in case of the cell cultures.Do the authors in vitro consider it will work?
The experimental animals have been used to obtain tissues of placenta and teratoma for experimental purpose.However, it can be avoided by contacting a bio-bank for tissue samples.The experimental animals, strictly, must be used only for developing or reproducing a pathogenesis model which must be used for final prevention and treatment study.
The reasons behind selection of placenta and teratoma tissues for study purpose, it needs to be discussed in details.Why it overruled other tissues?
Total number of animals used in this study, it needs to be written in section "Mice".We have read this submission.We believe that we have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however we have significant reservations, as outlined above.
Number of samples evaluated in each case, it requires to be increased to a minimum number to n=3 for statistically analyze the data.The figures are showing n=1, n=2 in most of the analyzed tissue samples.It requires marking well by arrows the findings mentioned in the paper in the figures.
requires marking well by arrows the findings mentioned in the paper in the figures.Is the work clearly and accurately presented and does it cite the current literature?YesIs the study design appropriate and is the work technically sound?Yes Are sufficient details of methods and analysis provided to allow replication by others?PartlyIf applicable, is the statistical analysis and its interpretation appropriate?Not applicableAre all the source data underlying the results available to ensure full reproducibility?YesAre the conclusions drawn adequately supported by the results?YesNo competing interests were disclosed.Competing Interests:Referee Expertise: The reviewers have expertise in handling retinal tissues and cells, and published similar articles describing methodologies for quenching autofluorescence.

Table 1 . Summary of treatments for immunofluorescence. Treatment Abbreviation and Chemical Formula
(Kuckenberg et al., 2010), detected with AlexFluo488 conjugated secondary antibody.The fluorescence signals at 488 nm (FITC),