P1 Does the degree of labeling have an effect on antibody affinity?

77 Pages·2017·1.53 MB·English

by  Ghost Writer

P1 Does the degree of labeling have an effect on antibody affinity?
Agnes Szabo (1), Timea Szatmari (2), Ildiko Radi (2), Laszlo Ujlaky-Nagy (2), Janos
Szollosi (3), Peter Nagy (2)
1) MTA-DE Cell Biology and Signaling Research Group, University of Debrecen, Egyetem square 1, Debrecen,
Hungary
2) Department of Biophysics and Cell Biology, University of Debrecen, Egyetem square 1, Debrecen, Hungary
3) MTA-DE Cell Biology and Signaling Research Group, University of Debrecen, Egyetem square 1, Debrecen,
Hungary and Department of Biophysics and Cell Biology, University of Debrecen, Egyetem square 1, Debrecen,
Hungary
Keywords: fluorescence antibody, DOL, fluorescence anisotropy, antibody affinity,
quantum yield
Abstract: Background: Antibodies conjugated to fluorescent dyes are widely used for
labeling cells. Since the degree of labeling (DOL, fluorescent dye/protein ratio) only gives
the mean number of fluorescent dyes conjugated to an antibody, some of the antibodies
contain fewer, while others contain more fluorescent dye/antibody. Methods: Monoclonal
antibodies were conjugated with AlexaFluor647 or AlexaFluor546. Cell surface receptors
were labeled with antibodies with different degrees of labeling (DOL ranging between 1
and 5). Fluorescence emission spectra were recorded in order to determine the relative
brightness of the antibodies with different degrees of labeling. In parallel experiments the
fluorescence intensity of cells labeled by the same antibodies was measured by flow
cytometry. The receptor-bound antibodies were immunoprecipitated by protein G and the
fluorescence anisotropy of the antibody stock solutions and the immunoprecipitated,
bound antibodies was measured. Results: The fluorescence intensity of the antibody
stock solutions did not increase proportionally to the DOL suggesting that fluorophores
undergo self-quenching. The intensity of the bound fraction did not change proportionally
to the intensity of the antibody stock when plotted as a function of the DOL suggesting
that the antibody affinity also decreases as a function of the DOL. Systematic differences
were observed between AlexaFluor546 and AlexaFluor647 in this regard. Fluorescence
anisotropy measurements confirmed these observations by revealing that the DOL of the
cell-bound antibody fraction was typically lower than that of the stock. Model calculations
confirmed that the distinct dependence of the quantum yield and the affinity on the DOL
can lead to the different behavior of the two dyes. Conclusions: The lower affinity of
antibodies with high DOL has to be kept in mind when selecting a fluorescent antibody or
when correcting fluorescence intensities for the degree of labeling.
P2 The dynamic of Tff1 and Tff3 peptide presence in mouse embryonic
gastrointestinal system from day 14 to 18 of intrauterine development
Nikola Bijelić (1), Tatjana Belovari (1), Ivana Lovrić (1), Mirela Baus Lončar (1)
1) Department of Histology and Embryology, Faculty of Medicine, University of Osijek, J. Huttlera 4, 31000
Osijek, Croatia
Keywords: trefoil factor, mouse, embryonic development, immunohistochemistry
Abstract: INTRODUCTION: Trefoil factor family (Tff) peptides are important for protection
and restitution of gastrointestinal (GI) mucosa. They are found in a large part of the GI
tract, from the oral cavity to the rectum, as well as in some glands associated with the GI
tract. Endodermal to gastrointestinal epithelium differentiation in mice occurs during 15th
or 16th day of intrauterine development. Tff peptides have been researched in this
context to an extent, but no systemic immunohistochemical studies have been performed
in order to describe the presence of these peptides depending on the developmental
stage. The aim of this research was to describe the presence of Tff1 and Tff3 peptide in
the GI mucosa from day 14 to 18 of mouse intrauterine development. MATERIALS AND
METHODS: 4% paraformaldehyde fixed and paraffin-embedded CD1 mouse embryos at
gestational days 14 to 18 were used. Sagittal sections 6μm thick were cut and transferred
to adhesive slides. The embryos were stained with purified proprietary, self-made anti-
Tff1 and anti-Tff3 rabbit primary polyclonal antibodies. Labeled Streptavidin Biotin (LSAB)
method was used, with PBS as a negative control. Mayer’s hematoxylin was used for
counterstaining. RESULTS: The localization of Tff1 and Tff3 peptide was similar, but
somewhat different from that of adult mice, with specific localization patterns for both
peptides. Immunohistochemistry showed weak or no signal at 14-day stage and
strenghthening of the signal until day 18. Both peptides were found in stomach, small
intestine, colon, liver and pancreas. Their localization and distribution depended on the
developmental stage. CONCLUSION: Tff1 and Tff3 peptides can be viewed as markers of
the GI mucosa maturation during embryonic development. They also might have an
influence on the differentiation of endoderm into mature epithelium. Further research
might confirm or deny that, as well as show the extent of their influence on embryonic
development of GI system.
References: 1. W. Hoffmann et al. Histol. Histopathol. 16 (2001) 319–334. 2. G. Regalo et al. Cell.
Mol. Life Sci. 62 (2005) 2910–2915. 3. T. Belovari et al. Bosn J Basic Med Sci. 15 (2015) 33–37.
Caption: Strong TFF3 immunohistochemical staining in the mucosa of mouse colon at day 18 of
embryonic development. Scale bar: 60 µm.
P3 A novel and economical staining method for quick identification and evaluation
of FFPE sections in a histology laboratory
Edi Rođak (1), Ivana Lovrić (1), Nikola Bijelić (1), Tatjana Belovari (1)
1) Department of Histology and Embryology, Faculty of Medicine, University of Osijek, J. Huttlera 4, 31000
Osijek, Croatia
Keywords: histology, staining and labeling, paraffin, haematoxylin
Abstract: Histological procedures can be a complex and lengthy process. Staining of
formalin-fixed, paraffin-embedded (FFPE) sections requires a lot of intermediate steps in
which the consumption of chemicals can be extensive and time-consuming. Furthermore,
an unknown tissue sample may occur, or a quick look at the tissue structure may be
required for further planning. In order to avoid the numerous intermediate steps, we
developed a quick method for staining of FFPE sections that takes about 10 to 15
minutes overall. Human liver, skin and small intestine FFPE samples from our
department’s histological archive were used. 5µm sections were cut, placed on slides and
dried for an hour on a thermal plate at 50 °C. Dry slides were left to cool down and 1.5 ml
3% (w/v) haematoxylin in 100% ethanol was added on slides using a micropipette. After 5
minutes, 1 ml of 2.3% iron (III) chloride solution was added to the already present
haematoxylin solution. Mixed solution was left on slides for another 5 min, then slides
were washed with dH2O. Slides were then immersed in same iron chloride solution to
remove excess stain and rinsed with dH2O. The slides were examined and photographed
within an hour after the staining was completed; no cover slip was placed on the tissue.
Blue nuances of haematoxylin were predominant, and paraffin crystals were visible.
Characteristic details could be identified in all three examined tissues (e.g. central veins,
hepatocytes, portal triads; epidermis, hair follicles, sebaceous glands; intestinal villi,
crypts, goblet cells). Although the paraffin crystals distorted the image to an extent, the
tissues could be easily identified, with characteristic tissue details visible enough for quick
analysis. This method can be useful for quick identification or screening of FFPE
samples. Apart from being fast, it is also valuable for economical reasons, since the
usage of xylene, ethanol and other chemicals is reduced to minimum.
References: The authors wish to thank Ms Danica Matić for her invaluable help in the histology lab.
Caption: A detail from the small intestine. Intestinal villi, crypts, goblet cells are easily
identifiable. Scale bar: 300µm.
P4 Localising fluorescence to improve correlative light and electron microscopy
workflows: ultraLM and miniLM
Elisabeth Brama (1), Christopher J. Peddie (1), Gary Wilkes (1), Yan Gu (1), Lucy M.
Collinson (1), Martin L. Jones (1)
1) The Francis Crick Institute, London, UK
Keywords: CLEM, in-resin fluorescence, locator tool, serial block face SEM, ultramicrotome
Abstract: The increase in availability of dual-contrast samples containing both
fluorescence markers and electron contrast aids the interpretation of macromolecular
function within the complex cellular landscape using correlative light electron microscopy.
Currently these samples are imaged using separate light and electron microscope or
dual-modality integrated microscopes. These samples also create an opportunity to use
the fluorescent signal to facilitate ‘smart’ automated image acquisition. We have
developed two new locator tools for finding and following fluorescent cells in-resin
fluorescence blocks. The ultraLM is a fluorescence microscope that integrates with an
ultramicrotome, which enables light imaging of the block face during sectioning. The
fluorescent signal is used to guide which sections are collected for subsequent
transmission electron microscopy or array tomography. The miniLM is a fluorescence
microscope that integrates with serial block face scanning electron microscopes, which
allows fluorescence guided image acquisition and provides the basis for developing
‘smart tracking’ of fluorescent structures during automated serial electron image
acquisition from large cell and tissue volumes. Both the ultraLM and the miniLM were
conceived and designed with the intention of retrofitting into existing systems to enable
the correlative imaging community to replicate these designs and integrate them into their
own platforms.
References: Brama, Elisabeth et al., “Wellcome open research 1 (2016): 26. PMC. Web. This work
was supported by the Francis Crick Institute which receives its core funding from the Wellcome
Trust [FC001999]; Cancer Research UK [FC001999]; the UK Medical Research Council
[FC001999]; and from the UK Medical Research Council, BBSRC and EPSRC under grant
[MR/K01580X/1]
Caption: Proof-of-principle ultraLM operation was demonstrated using IRF blocks containing HeLa
cells expressing GFP-H2B.
P5 Dynamic imaging of plant cortical microtubules using structured illumination
microscopy
O. Šamajová (1), G. Komis (1), M. Mistrik (2), M. Ovečka (1), J. Bartek (2), J. Šamaj (3)
1) Centre of the Region Haná for Biotechnological and Agricultural Research, Palacký University, Olomouc,
Czech Republic
2) Institute of Molecular and Translational Medicine, Palacký University, Olomouc, Czech Republic
3) Genome Integrity Unit, Danish Cancer Society Research Center, Copenhagen, Denmark
Keywords: cytoskeleton, microtubules, plant, super-resolution microscopy, SIM
Abstract: The use of time-lapse structured illumination microscopy (SIM) imaging in plant
cells considerably enhanced spatial resolution of cortical microtubules. Here we describe
dynamic SIM imaging of cortical microtubule organization and dynamics in cells of diverse
plant tissues. As a proof of principle we explore the potential of SIM to elucidate
microtubule size, bundling and de novo branched formation. We also characterized
quantitative parameters of plus end associated dynamic instability events at subdiffraction
resolution in Arabidopsis seedlings expressing two microtubule markers GFP-MBD and
GFP-TUA6 (Komis et al. 2014). Time-lapse SIM imaging allowed the visualization of
subdiffraction, length changes of the microtubule plus end, and dynamic instability
behavior of both plus and minus ends during free, intrabundle, or microtubule-templated
microtubule growth and shrinkage. Conclusively, the present work shows the capacity of
SIM for superresolution time-lapse plant cell imaging, revealing unprecedented details of
dynamic microtubule organization.
References: George Komis et al., Plant Physiol. 165 (2014) 129-148. This work was supported by
the grant No. LO1204 (Sustainable development of research in the Centre of the Region Haná)
from the National Program of Sustainability I, MEYS.
P6 Light sheet microscopy of tissue-specific and developmentally-regulated
nuclear localization of END BINDING protein 1c in root tip cells of Arabidopsis
thaliana
M. Ovečka (1), D. Novák (1), A. Kuchařová (1), G. Komis (1), J. Šamaj (1)
1) Department of Cell Biology, Centre of the Region Haná for Biotechnological and Agricultural Research,
Palacký University Olomouc, Olomouc 783 71, Czech Republic
Keywords: development, end-binding 1c (EB1c), light sheet microscopy, nucleus, root apex
Abstract: END BINDING protein 1c (EB1c) is a plant-specific protein binding mitotic
microtubules, while it is localized in nuclei of non-dividing cells of root apex. Due to this
specific localization pattern, EB1c has been previously documented exclusively in dividing
cells and its possible roles were proposed only in the context of plant meristematic
activity. However, tissue- and organ-specific localization in the developmental context
must be addressed to better understand EB1c function in root development. Appropriate
imaging techniques are necessary for developmental long-term imaging, allowing plants
to grow under nearly environmental conditions inside of microscopic chamber (Ovečka et
al., 2015). Therefore, we quantified the developmentally regulated nuclear levels of GFP-
tagged EB1c protein under the control of native EB1c promoter using advanced and
minimally invasive light sheet microscopy (Fig. 1). We correlated in detail nuclear size
with EB1c expression levels in diverse tissues like epidermis, cortex and endodermis, and
in different developmental zones including meristematic, transition and elongation zone of
Arabidopsis thaliana roots (Novák et al., 2016). Our results demonstrate a high potential
of light sheet microscopy in multidimensional live cell imaging of plant root development.
Obtained spatial and temporal resolution allowed to monitor developmental cell
reprogramming during the transition of root cells from cell proliferation to cell
differentiation.
References: 1. M. Ovečka et al., Nat. Protoc. 10 (2015) 1234-1247. 2. D. Novák et al., Front. Plant
Sci. 6:1187 (2016). This work was supported by the Czech Science Foundation (GACR) grant 14-
27598P, by grant no. LO1204 (Sustainable development of research in the Centre of the Region
Haná) from the National Program of Sustainability I, Ministry of Education, Youth and Sports, Czech
Republic and by student project IGA_PrF_2016_012.