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Immunofluorescence staining of paraffin sections: creating DAB staining like virtual digital images using CMYK color conversion

  • RE Kavetsky Institute of experimental pathology

Abstract and Figures

Crystal violet treatment of formalin fixed paraffin embedded tissue slides greatly reduces the endogenous autofluorescence, and allows immunofluorescence (IF) staining with FITC or Alexa488 conjugated antibodies. Using cold CCD camera to capture the fluorescence images makes this staining method very sensitive. Here we show that combination of IF with the simultaneous recording of crystal violet induced red and Hoechst 33258 induced blue fluorescence permits the localization of the IF signal over a cytoplasmic: nuclear red:blue stain that visualizes the microscopic anatomy of the underlying tissue. To make the visual interpretation of the IF staining easier for microscopists, who are used to DAB staining over weak hematoxilin-eosin background, we created a simple color conversion procedure that turns the captured three-color fluorescence RGB (red, green, blue) images over a black background into four color CMYK (cyan, magenta, yellow, key color (black)) images.
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Experimental Oncology 30, 327–329, 2008 (December) 327
Immunostaining of formalin fixed and paraffin em-
bedded sections is routinely carried out using enzyme
conjugated secondary antibodies such as alkaline
phosphatase or horse radish peroxidase (HPR). These
enzymes produce insoluble chromogenic products
from soluble colorless substrates that are deposited at
the site of antigen:antibody complex. A frequently used
substrate of HPR is diaminobenzidinetetrahydrochlo-
ride (DAB) that in combination with different metal ions
such as silver or nickel produces dark brown or black
deposits. The underlying tissue structures are regularly
visualized by a weak hematoxylin or hematoxylin-eosin
(HE) counterstaining. The resultant images can only be
captured using color cameras. Due to the mixture of
colors and the nonlinear nature of the dye deposition
it is rather difficult to quantify the amount of antigen
using this technique. Fluorescent dye conjugated
secondary antibodies can overcome this problem by
allowing monochromatic capturing of images over
the dark background using highly sensitive cold CCD
cameras. The signal amplification through double layer
immunostaining with fluorochrome conjugated se-
condary antibodies and the image acquisition through
black and white cameras allow a highly linear quantita-
tion of the antigen. The greatest hinder for using im-
munofluorescence staining on formalin fixed paraffin
embedded sections is the relatively high greenish
autofluorescence that interferes with the most fre-
quently used dyes such as Fluorescein isothiocyanate
(FITC). The strength of autofluorescence among
other factors is dependent on the tissue type, quality
of fixative, time of fixation, thickness of the sections
and the wavelength of excitation light [1]. The extent
of interference between the specific immunostaining
and background fluorescence can be controlled by
several different means. One of the simplest methods
is to use carefully selected high affinity antibodies in
combination with good quality secondary antibodies to
detect relatively abundant antigens and carry out the
imaging in narrow focal planes using laser scanning
confocal microscopy [2]. Alternative ways to decrease
the signal to noise ration are: signal amplification by
tyramid deposition [3, 4], using fluorescent dyes that
has red emission [5], digital subtraction of separately
measured background fluorescence [6] or use of
spectral imaging in combination with linear unmixing.
Alternatively the slides can be treated with different
quenching agents, such as sodium borohydride fol-
lowed glycine treatment [6], Sudan black ammonia
[7] or Crystal violet [8–12]. This latter even allows
quantitation of relatively weak signals such as prolife-
rating cell nuclear antigen (PCNA) [13] or quantitative
detection of apoptosis on highly autofluorescent liver
sections [14].
In the present paper we tried to exploit the feature
of the Crystal violet staining that creates a strong red
fluorescence of all cellular structures when illuminated
at 560 nm. Here we show that using this dye it is pos-
sible to visualize the underlying tissue architecture.
When combined with DNA staining dyes such as
Hoechst 33258 or DAPI this method can reveal very
detailed cellular morphology. To demonstrate the
feasibility oft the combination of immunofluorescence
staining with Crystal violet: DAPI counterstaining
we have carried out IF detection of p53 in the nuclei
of neoplastic endometrium. 5 µm thick slides were
deparaffinized in xylol and rehydrated in descending
ethanol series. The epitopes were recovered by boiling
L. Buchynska1, *, E. Kashuba2, 3, L. Szekely2, 3
1Institute of Experimental Pathology, Oncology and Radiobiology NAS of Ukraine, Kiev 03022, Ukraine
2Department of Microbiology and Tumor and Cell Biology, Karolinska Institute, Stockholm S17177, Sweden
3Center for Integrative Recognition in the Immune System, Karolinska Institute, Stockholm S17177, Sweden
Crystal violet treatment of formalin fixed paraffin embedded tissue slides greatly reduces the endogenous autofluorescence, and
allows immunofluorescence (IF) staining with FITC or Alexa488 conjugated antibodies. Using cold CCD camera to capture the
fluorescence images makes this staining method very sensitive. Here we show that combination of IF with the simultaneous recording
of crystal violet induced red and Hoechst 33258 induced blue fluorescence permits the localization of the IF signal over a cyto-
plasmic: nuclear red:blue stain that visualizes the microscopic anatomy of the underlying tissue. To make the visual interpretation
of the IF staining easier for microscopists, who are used to DAB staining over weak hematoxilin-eosin background, we created
a simple color conversion procedure that turns the captured three-color fluorescence RGB (red, green, blue) images over a black
background into four color CMYK (cyan, magenta, yellow, key color (black)) images.
Key Words: immunofluorescence microscopy, paraffin sections, digital imaging.
Received: November 10, 2008.
*Correspondence: Fax: +38 (044) 258-16-56
Abbreviations used: CMYC — cyan, magenta, yellow, key color;
DAB — diaminobenzidinetetrahydrochloride; FITC — fluorescein
isothiocyanate; HE — hematoxylin-eosin; HPR — horse radish per-
oxidase; IF — immunofluorescence; RGB — red, green, blue.
Exp Oncol 2008
30, 4, 327–329 TEChNICAL ADVANCE
328 Experimental Oncology 30, 327–329, 2008 (December)
the slides in 100 mM citrate buffer in microwave oven
for 5 min. We used DO7 anti-p53 mouse monoclonal
antibodies in combination with FITC conjugated rab-
bit anti-mouse secondary antibody (both from DAKO,
Carlstrup, Denmark). The antibodies were diluted in the
following blocking buffer: 0.2% Tween 20, 5% glycerol,
and 3% BSA in PBS. Following the immunostai ning the
slides were treated with 0.5% Crystal violet in 0.9%
NaCl (freshly diluted from 10% stock in methanol) for
10 min. After washing 3 times in 0.9% NaCl the slides
were rinsed in PBS and mounted with 80% glycerol and
2.5% DABCO (pH 8.5). The images were visualized in
a Leitz fluorescence microscope and were captured
with a Hamamatsu C4880 CCD camera. We found
that using RGB color-coding the green immunofluo-
rescence signal can be conveniently overlayed on the
red and blue cellular background. The drawback of
this method is that the resultant images are on black
background and do not resemble the color experience
of conventional immunostaining. Here we show that
a simple image conversion procedure can turn the
original RGB ima ge into a white background image that
resembles a hematoxylin-eosin staining combined with
black immunostaining deposits. The procedure that is
summarized in Fig. 1 is the following:
1. An empty image with white background is created
in CMYK mode in identical size to the RGB image.
2. The content of the red (R) channel is copied to
the magenta (M) channel of the CMYK image.
3. The content of the green (G) channel is copied
to the CMYK black (K) channel.
4. The content of the blue (B) channel is copied to
the CMYK cyan (C) channel.
5. All channels of the CMYK image are selected
and inverted.
6. The content of the yellow (Y) channel is de-
The advantage of the above described three color
staining method is that fluorescent images that were
captured by monohrome CCD cameras over a black
background can be visualized in a way that is similar to
the DAB enhanced immunostaining of routine immuno-
histology. The individual components (red cytoplasmic,
blue nuclear and black immunostaining) of the resul-
tant image are kept as separate information and can be
freely toggled, both in the RGB, or in the CMYK mode,
by switching on and off the corresponding channels
as illustrated on Fig. 2. Importantly, the signal intensi-
ties of individual cells in the separate channels can be
digitally quantitated with a 12 bit broad dynamic range.
The described method also permits visualization of
double immunostaining results obtained using e.g.
FITC and Texas Red signals on sodium borohydride
treated slides. In this case the crystal violet pattern in
the red channel is replaced with the specific second
(red) immunostaining. The resultant image shows the
two specific antigens in black and red, respectively,
over a blue nuclear background similarly to HRP and
alkaline phosphatase double staining.
Copy to
Fig. 1. Digital conversion of immunofluorescent RGB image
into a “DAB combined with hematoxylin:eosin” like image. The
example for RGB image is a section of a human displastic en-
dometrium stained for p53 using FITC conjugated secondary
antibody (green) with the fluorescence of the green autofluo-
rescence quencher crystal violet (red) and nuclear staining with
Hoechst 33258 (blue). The content of the red, green, and blue
channels of the RGB image are copied into an identical size CMYK
image, into the magenta, black and cyan channels respectively.
The content of the CMYK channels are inverted and the yellow
channel is deleted
Immunostaining HE like background Overlap
Fig. 2. The digitally created “DAB/HE” like image allows the
separate presentation of the specific immunostaining (black)
and the underlying histology (red/blue) as well as the combina-
tion of the two
Thus, we have developed an imaging method that
allows vitalic in silico visualization of imunofluores-
cence signal on paraffin embedded slides in a manner
similar to the conventional immunoperoxidase stain-
ing. The signal is quantifiable over a 4096 gray scale
level. Moreover it can be freely toggled over an HE like
nuclear:cytoplasmic background.
This study was supported by the Swedish Can-
cer Society (Cancerfonden), the Swedish Reseacrh
Council and by Swedish Institute in the frame of Visby
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Окраска кристаллическим фиолетовым парафиновых срезов, полученных из ткани, фиксированной в формалине, значительно
уменьшает явления аутофлуоресценции и обеспечивает иммунофлуоресцентную (ИФ) окраску антителами, конъюгированны-
ми с FITC или Aleхa-488. Использование CCD-камер для регистрации флуоресцентных изображений делает этот метод очень
чувствительным. Наша цель — разработать метод трансформации RGB (red, green, blue) — флуоресцентных изображений в
CMYC (cyan, magenta, yellow, key color (black) ) изображения. Показано, что комбинация ИФ с одновременной регистрацией
красной и голубой флуоресценции, индуцированной соответственно кристаллическим фиолетовым и Hoechst 33258, позволяет
определять ИФ-сигнал как цитоплазматично-ядерное красно-голубое окрашивание, которое визуализирует морфологические
особенности прилегающей ткани. Для упрощения интерпретации ИФ-окраски патологами, привыкшими к окрашиванию
ДАБ на фоне гематоксилин-эозина, нами была создана технология простого цветового перехода, который превращает за-
регистрированные трехцветные RGB-флуоресцентные изображения на черном фоне в четырехцветные CMYC-изображения
на белом фоне, используя программы работы с изображениями.
Ключевые слова: иммунофлуоресцентная микроскопия, парафиновые срезы, цифровые изображения.
Copyright © Experimental Oncology, 2008
... analysis were performed, as described earlier 32 . Staining was evaluated manually, counting the PKN1-positive EC cells. ...
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The method described allows staining of juxtaglomerular granules in a deep purple colour against faint purple background and deep purple erythrocytes. Paraffin sections of mouse kidney fixed in 10% formalin buffered with phosphate are brought to water, stained 3 min in 0.01% crystal violet, rinsed in tap water (or stained 3 min in 0.1% crystal violet in NaHCO3, rinsed in 1% NaHCO3), blotted, differentiated in a 1 : 1 aniline-xylene mixture, cleared in xylene and mounted in a resin.
A combination of methods for fixation (sublimate, cobalt nitrate, formaldehyde, acetic acid in water), inclusion (celloidin dissolved in methyl salicylate, paraffin-paraplast) and staining was used to make serial sections easy, to avoid clefts and to give a good picture of segmentation mitoses, as well as a good contrast of yolk and cytoplasmic components. Four methods of staining were used concerning the Urodele eggs: Safranin-methyl blue-orange G, safranin-picro-blue black naphthol (Curtis), safranin-violet crystal-orange G (Flemming) and Feulgen-methyl blue-orange G. The achromatic apparatus of the normal segmentation mitoses is clearly delineated and the relationships between astral fibers and yolk are different at metaphase and anaphase. By these methods, particularly suitable for demonstration of nuclei, cytoplasm and achromatic apparatus, the cleaving egg may be used as a test for the inhibition of achromatic apparatus and chromosome damage by antimitotic substances. The contrast between vitelline cytoplasm and cytoplasmic non-vitelline abnormal fibrillar systems, produced by transformation of astral and diastematic fibres, is made particularly evident by these methods of staining.
A saturated solution of orseillin BB in 3% acetic acid followed by a 1% aqueous solution of crystal violet provides an excellent differential staining for sections of ascomycetous fructifications. The technique stains a wide variety of fungus and host cells, revealing considerable morphological and cytological detail. It is appropriate for microscope slides both of unfixed material mounted in water and of picric acid-fixed paraffin sections.
WILSON's crystal violet method stains not only the granules of juxtaglomerular cells but also red blood cells, myelinated tracts in the C.N.S., striated muscle, the secretion granules of sweat glands and the intercellular bridges of the epidermis. The purpose of this paper is to ascertain whether WILSON's stain can demonstrate, in paraffin embedded sections, proteolipids and/or lipoproteins; and to determine the histochemical specificity of the stain. The material chosen was central and peripheral myelin, (rats, human) since this is known to contain trypsin resistant, tryptophan containing proteolipids. A modification of this staining for renin granules shows both the central and peripheral nerve fibers on paraffin sections with reproducible results. Fixation, pH and temperature are critical. This stain is considered to demonstrate nerve fibers on account of its capacity to combine with proteolipids.
The primary fluorescence (autofluorescence) of some cell and tissue components depends on the fixative and fixing time, as well as on the thickness of paraffin sections and the wavelength of exciting light. The highest autofluorescence emission (pale green) was observed by using violet-blue excitation. After aldehyde fixation, the autofluorescence of some tissue structures was higher than after methanol or ethanolacetic acid. These features must be taken into account when fluorescence microscopy is applied to the study of cell smears and paraffin embedded tissues after flurochroming or immunofluorescence reactions.
The evaluation of apoptosis is an important aspect in the study of chemical carcinogenesis. Methods were developed employing the ApopTag kit (Oncor, Gaithersburg, MD) and automated image analysis to quantitate the distribution of apoptosis in formalin-fixed, paraffin-embedded liver tumor sections from rats induced by 2-acetylaminofluorene. Specific treatments of tissue sections were developed that permitted quenching of background tissue autofluorescence with crystal violet and permitted permeating the fixed tissue sections by trypsin digestion. Tissue sections were stained by using the ApopTag kit for detection of in situ apoptosis and with propidium iodide as a counterstain for tissue nuclei. Automated statistical evaluation of the percentage of tissue nuclei also staining positively for apoptosis was determined by using dual fluorescence detection and imaging laser microscopy. The quantitative results indicated that the staining index for apoptosis in normal liver was 0.14 +/- 0.04% whereas well and poorly differentiated tumors showed increases of 3.48 +/- 0.59% and 7.41 +/- 0.81%, respectively. The staining indexes for apoptosis showed a tight correlation between fluorescent and peroxidase-diaminobenzidine detection in sequential tissue sections. The use of in situ apoptosis staining and automated image analysis for rapid identification and quantitation of cells undergoing death in fixed tissue will expedite additional studies to evaluate the in situ tissue pathobiology of tumors and aid in the study of molecular mechanisms associated with cancer development and treatment.
Scanning laser cytometric analysis of fluorochrome-labeled cells was used to survey and quantitate the distribution of proliferating cells in formalin-fixed, paraffin-embedded tissue sections from hyperplastic and neoplastic rat livers. The technique used fluorescent immunochemical staining of proliferating cell nuclear antigen (PCNA) as a marker for proliferating cells and propidium iodide as a fluorescent nuclear counterstain. Use of an antigen retrieval treatment improved detection of PCNA and treatment of tissue sections with crystal violet improved the sensitivity of the method by quenching background autofluorescence. PCNA evaluation of cell proliferation in regenerating rat liver 0-48 h post-partial hepatectomy showed that 3-43% of cells stained positively for PCNA, a pattern closely correlating with previously reported rates of maximum DNA synthesis. The PCNA staining patterns observed among cells in neoplastic nodules were more focal in distribution and indicated that from 5 to 25% of the cell nuclei per nodular region stained positively for PCNA. This use of image analysis for the rapid identification of proliferating cell areas in fixed, paraffin-embedded tissue active in neoplastic growth will expedite in situ cytochemical and molecular studies attempting to identify key differences between hyperplastic and neoplastic growth.