New fluorescent Fluolid dyes have advantages over others such as stability against heat, dryness, and excess light. Here, we performed simultaneous immunostaining of renal tumors, clear cell renal cell carcinoma (RCC), papillary RCC, chromophobe RCC, acquired cystic disease-associated RCC (ACD-RCC), and renal angiomyolipoma (AML), with primary antibodies against Kank1, cytokeratin 7 (CK7), and CD10, which were detected with secondary antibodies labeled with Fluolid-Orange, Fluolid-Green, and Alexa Fluor 647, respectively. Kank1 was stained in normal renal tubules, papillary RCC, and ACD-RCC, and weakly or negatively in all other tumors. CK7 was positive in normal renal tubules, papillary RCC, and ACD-RCC. In contrast, CD10 was expressed in renal tubules and clear cell RCC, papillary RCC, AML, and AC-RCC, and weakly in chromophobe RCC. These results may contribute to differentiating renal tumors and subtypes of RCCs. We also examined the stability of fluorescence and found that fluorescent images of Fluolid dyes were identical between a tissue section and the same section after it was stored for almost three years at room temperature. This indicates that tissue sections can be stored at room temperature for a relatively long time after they are stained with multiple fluorescent markers, which could open a door for pathological diagnostics.
Owing to the increased availability of diagnostic markers for pathological evaluation of cancer, there has been an increased demand for staining valuable specimens with multiple and combinational markers. There have been approaches based on double, triple, and even quadruple staining of specimens with the respective numbers of markers [
Kidney and urinary tract cancers accounted for 8,334 deaths in 2012 in Japan, roughly 2% of all cancers [
In order to develop a new technique for immunohistochemical staining in the pathological diagnosis of cancer, we examined here tissue sections containing human renal tumors by means of quadruple staining using antibodies labeled with two Fluolid dyes, Fluolid-Green and Fluolid-Orange, in combination with Alexa Fluor 647 and 4′,6-diamidino-2-phenylindole (DAPI). Antibodies against Kank1, cytokeratin 7 (CK7), and CD10 proteins were used as the primary antibodies and Fluolid-conjugated IgG (Kank1 and CK7) and Alexa Fluor 647-conjugated IgG (CD10) were used as the secondary antibodies to detect the primary antibodies. The gene for Kank1 (
A rabbit anti-human cytokeratin 7 (CK7) antibody was purchased from Funakoshi (Tokyo, Japan), a goat anti-human neprilysin (CD10) antibody from R&D Systems (Minneapolis, MN), and donkey anti-mouse IgG and donkey anti-rabbit IgG from Jackson ImmunoResearch (West Grove, PA). Alexa Fluor 647-conjugated donkey anti-goat IgG was purchased from Life Technologies (Carlsbad, CA). Fluolid-Orange and Fluolid-Green were purchased from Cosmo Bio (Tokyo, Japan).
IgG was labeled with each Fluolid dye using a Fluolid-W protein/antibody labeling kit (International Science Technology, Fukuoka, Japan) according to the manufacturer’s instructions. Briefly, IgG was dissolved into 0.2 M sodium bicarbonate buffer (pH 8.3), mixed with a Fluolid dye (dissolved in DMSO), and incubated for 2 hr at room temperature. Unreacted dye was removed with a NAP-5 column (GE Healthcare Japan, Tokyo, Japan).
Paraffin-embedded specimens from RCC patients were obtained upon written informed consent after approval from the ethics committees of Tokyo Medical University and the National Institute of Advanced Industrial Science and Technology. Specimens were deparaffinized by washing three times with fresh xylene for 5 min and then washing with graded ethanol, followed by washing twice with phosphate-buffered saline (PBS). For retrieving antigens, samples were boiled in 10 mM citrate buffer (pH 6.0) for 10 min using a microwave oven as reported previously [
To examine the usefulness of Fluolid dyes in multiple immunostaining, we performed immunostaining using antibodies labeled with two different Fluolid dyes (Fluolid-Green and Fluolid-Orange). Alexa Fluor 647 was used as the third fluorescent dye and also as a reference. Antibodies against Kank1, CK7, or CD10 proteins raised in mice, rabbits, or goats, respectively, were used as the primary antibodies and fluorescently labeled IgGs for respective species were used as the secondary antibodies. DAPI was included as the fourth dye to localize nuclei. We first performed single-dye immunostaining of kidney tissues to detect Kank1, CK7, or CD10, respectively, and confirmed that the respective fluorescence images did not overlap (data not shown). We then immunostained normal mouse kidney tissues with multiple markers (data not shown) and then applied the system to immunostain human renal tumors.
The results of simultaneous staining of renal tumors with multiple markers labeled with Fluolid dyes are shown in Figure
Summary of immunostaining of Kank1, CK7, and CD10 in renal tumors.
Marker | Renal tumor | ||||
---|---|---|---|---|---|
Clear cell RCC | Papillary RCC | Chromophobe RCC | AML | ACD-RCC | |
Kank1 | − | + | − | − | + |
CK7 | − | ++ | − | − | + |
CD10 | ++ | + | ± | + | + |
Immunostaining of renal tumors. Antibodies against Kank1 (
We also examined the stability of Fluolid dyes in a section that was stored at room temperature for almost three years (Figure
Stability of fluorescent dyes in histopathological sections. Fluorescent images were examined for a tissue section before (a) or after (b) the storage at room temperature for almost three years. A normal human kidney tissue section was subjected to quadruple staining with three different Fluolid dyes and DAPI. Antibodies against Kank1, CK7, and CD10 were used as the primary antibodies, and Fluolid-Green-conjugated anti-mouse IgG, Fluolid-Orange-conjugated anti-rabbit IgG, and Alexa Fluor 647-conjugated anti-goat IgG were used as the secondary antibodies. Bar = 20
In this study, we examined whether Fluolid dyes are useful for pathological diagnosis and showed examples of quadruple staining of human renal tumors using Fluolid dyes (Fluolid-Green and Fluolid-Orange) along with Alexa Fluor 647 and DAPI. Antibodies against Kank1, CK7, and CD10 were used as the primary antibodies and fluorescently labeled IgGs were used as the secondary antibodies. To effectively separate the images from different fluorescent dyes, it was necessary to set up an appropriate set of fluorescence filters. We first examined the assay system including the filter set by evaluating whether the images were completely separated, observing the images after single, double, or triple staining using all combinations of Fluolid dyes, Alexa Fluor 647, and DAPI (see Section
Using this system, we performed quadruple staining of clear cell RCC, papillary RCC, chromophobe RCC, AML, and ACD-RCC (Figure
As summarized in Table
CD10 is a surface glycoprotein identified in a variety of healthy cells, where it hydrolyzes peptide bonds and decreases the cellular response to local peptide hormones. In the normal kidney, CD10 is strongly expressed at proximal tubular cell brush borders [
We also examined the stability of fluorescently labeled markers on a section that was stored at room temperature for almost three years (Figure
Multiple immunoenzyme staining systems have been developed by modifying standard chromogen-based immunoenzyme techniques. Double staining systems were developed to achieve maximum color contrast between red (such as 3-amino-9-ethyl-carbazole) and brown (such as 3,3′-diaminobenzidine or DAB) by unmixing spectral images [
In this study, we provided examples of quadruple staining of sections of human renal tumors using Fluolid dyes, Alexa Fluor 647, and DAPI. In conclusion, immunostaining with multiple fluorescently labeled markers will improve the classification of renal tumor subtypes using limited amounts of samples, which may be of particular help for pathologists and clinicians. Furthermore, Fluolid dyes will enable long-term preservation of stained histopathological sections. Hopefully, this multiple staining method will improve the accuracy of the diagnosis of various diseases.
The authors declare that there is no conflict of interests.
Dilibaier Wuxiuer and Yun Zhu contributed equally to this paper.
This research was supported partly by a Special Coordination Fund for Promoting Science and Technology (Encouraging Development of Strategic Research Centers), and a Knowledge Cluster Initiative program and a Grant-in-aid for Basic Areas from the Ministry of Education, Culture, Sports, Science and Technology of Japan.