collected and fixed, and 5 µμm thick sections were prepared for histological analysis. Human breast adenocarcinoma cells (MCF-‐‑7), human epidermal keratinocytes (HaCat) and human dermal fibroblasts were cultured as explained in publication I. UV-‐‑exposed skin samples for study II were gathered during the study of Kumlin et al. (Kumlin et al. 1998). The protocol for the preparation of the histological samples from skin is presented in study II.
The tumor samples for study III were collected in Oulu University hospital.
4.2 METHODS
4.2.1 EGFP-‐‑human Has1, 2, and 3 plasmid construction and transfection
The preparation of the human HAS1, 2, and 3 constructs is detailed in study I. Shortly, the open reading frame of each HAS was taken from human cDNA and amplified. The amplified HAS open reading frames were cloned to pEGFP-‐‑C1 vector. These plasmids were used for the transfections. The transfections were performed on cells cultured in 8-‐‑chamber slides (Ibidi GmbH, Martinsried, Germany) for microscopy and in 24-‐‑well plates (CELL STAR®, Greiner Bio-‐‑One, Kremsmunster, Austria) for the measurements of the hyaluronan concentrations in the growth medium.
4.2.2 Immunostaining
Live cells with the GFP-‐‑tagged HAS constructs were observed with a confocal microscope.
Cultured cells were fixed for the immunostainigs with 4% paraformaldehyde for 1 h in room temperature and permeabilized for 20 min with 1% BSA containing 0.1% Triton X100.
The cells were incubated overnight at 4°C with the HAS antibodies (Table 2), then washed and incubated with a fluorescein-‐‑labeled anti-‐‑goat secondary antibody. To perform double stainings for endoplamic reticulum, Golgi or hyaluronan, the HAS antibody was mixed with anti-‐‑Calnexin or anti-‐‑Golgi antibody, or bHABC (Table 2). The anti-‐‑ER and Golgi antibodies were visualized with a Texas Red anti-‐‑mouse secondary antibody, and hyaluronan with fluorescently labelled streptavidin. Biotinylated anti-‐‑goat secondary antibody (1:1000) with the avidin-‐‑biotin-‐‑peroxidase method was used for the wide-‐‑field microscopy.
HAS1, 2, and 3 immunostaining were used in the studies I, II and III. The tissue sections were incubated in 10 mM citrate buffer for 15 min at 120 °C in a pressure cooker. Non-‐‑
specific binding was blocked by incubation with 1% BSA and 0.1% gelatin in a phosphate buffer for 30 min. The sections were incubated overnight at 4°C with the hyaluronan synthase antibodies and biotinylated secondary antibodies and the avidin-‐‑biotin-‐‑
peroxidase method were used for their microscopic detection. Hyaluronan staining was used in studies I, II, and III. A complex containing the hyaluronan binding region of bovine articular cartilage aggrecan G1 domain and link protein (HABC) was biotinylated (bHABC)
18
and used as a probe to detect hyaluronan (Tammi et al. 1994b). The immunostaining for CD44 was done in studies II and III. The tissue sections were first incubated in an antigen retrieval solution (Dako, Carpentia, CA, USA) for 30 min at 95°C (II) and then with an anti-‐‑
CD44 antibody (IM7) overnight at 4°C. There was no retrieval treatment in study III for the detection of CD44 with the Hermes 3 antibody. The blocking solution for CD44 stainings was 1% BSA in a phosphate buffer. The bound primary antibody was detected using a biotin-‐‑labeled secondary antibody and the avidin-‐‑biotin-‐‑peroxidase method. The antibodies and probes used are listed in Table 2.
The specificity of the HABC-‐‑probe was controlled by pretreating the samples with hyaluronidase or by blocking the probe with hyaluronan oligosaccharides. The antibodies for the HASs were controlled with several methods. The HAS-‐‑antibodies were omitted and replaced with non-‐‑immune goat IgG from incubation, and the antibodies were also treated with the peptides used in the immunization. Possible cross-‐‑reactivities of the HAS-‐‑
antibodies against other HAS family members were tested in transfected cells overexpressing different HASs (I, Figure 6). In controls for the CD44 stainings the primary antibodies IM7 (study II) and Hermes 3 (III) were omitted
Table 2 The antibodies and the probe used in the thesis
Calnexin 1:100 Cell Signaling Technology,
Inc., Boston, MA, USA
Golgin-‐‑97 1:100 Molecular Probes, Eugene,
OR, USA
Biotinylated anti-‐‑goat 1:1000 Vector Laboratories,
Burlingame, CA, USA
Biotinylated anti-‐‑mouse 1:100 Vector Laboratories,
Burlingame, CA, USA
Biotinylated anti-‐‑rat 1:100 Vector Laboratories,
Burlingame, CA, USA
Fluorescein anti-‐‑mouse 1:1000 Vector Laboratories,
Burlingame, CA, USA
Texas Red anti-‐‑mouse 1:1000 Vector Laboratories,
Burlingame, CA, USA
19 4.2.3 Microscopy
In study I the fluorescent images were obtained by a Zeiss Axio Observer inverted microscope equipped with a Zeiss LSM 700 confocal module (Carl Zeiss Microimaging GmbH, Jena, Germany). A conventional light microscope (Zeiss Axio Imager.M2 light microscope, Carl Zeiss) was used in studies I, II and III.
4.2.4 Analysis of hyaluronan concentration
Hyaluronan secretion into the culture medium (I) was measured with an ELSA assay as previously described (Hiltunen et al. 2002). Shortly, 96-‐‑well plates were precoated with HABC. The diluted samples and the hyaluronan standards were incubated in the wells for 1h. The hyaluronan attached was quantified with the sequential incubations of biotinylated HABC, horse radish streptavidin and the substrate-‐‑chromogen solutions containing 0.01 % of 3,3′,5,5′-‐‑tetramethybenzidine and 0.005 % H2O2 in a 0.1 M sodium acetate-‐‑1.5 mM citric acid buffer.
4.2.5 Evaluation and statistical analysis
The intensity of the HAS1-‐‑3, hyaluronan and CD44 stainings were analyzed by two independent evaluators in studies II and III. The statistical analysis was performed using the SPSS program (IBM Corporation, Armonk, New York, USA). The comparison of the extent and intensity of the stainings between treatments was done with the Kruskal-‐‑Wallis and Mann-‐‑Whitney U-‐‑tests (II). The correlations between the amount of epidermal hyperplasia and the hyaluronan and CD44 parameters were determined with Kendall’s test (II). In study III the differences between mesotheliomas and adenocarcinomas were calculated with the Pearson chi-‐‑square tests.
20