Neutral buffered formalin

The RP specimens were fixed in 15% neutral-buffered formalin (Wako Pure Chemical Industries, Ltd., Osaka, Japan) for 48–96 h. Whole-organ prostate specimens were serially sectioned perpendicular to the rectal surface at 5-mm intervals. The specimens were embedded in paraffin, cut into 5-μm sections, and stained with hematoxylin and eosin.
Immunohistochemical staining for all specimens was carried out using monoclonal antibodies for chromogranin A. It was done on 4-μm-thick paraffin sections cut from the formalin-fixed tissues. Heat-induced epitope retrieval was performed in Tris-EDTA buffer (10 mM Tris Base, 1 mM EDTA solution, 0.05% Tween 20, pH 9.0). The sections were then incubated in 3% H₂O₂ for 10 minutes to eliminate endogenous peroxidase activity. The primary rabbit anti-chromogranin A polyclonal antibody (1 : 100, Dako, Glostrup, Denmark) and horseradish peroxidase-conjugated anti-rabbit secondary antibody were used for 60 minutes and 30 minutes at room temperature, respectively. Color development was accomplished with 3,3′-diaminobenzidine. The nuclei were then counterstained with hematoxylin. Only manifest cytoplasmic staining was defined as a positive reaction. Negative controls were incubated with normal rabbit serum instead of the polyclonal antibody.

Lungs and spines from mice were fixed in 10% neutral buffered formalin (Wako Pure Chemical Industries, Ltd., Osaka, Japan), processed, embedded in paraffin, sectioned at 5-6 μm, stained with H&E, and examined and photographed under a microscope. The spines were decalcified using decalcifying solution A (Plank Rychlo Method, Wako Pure Chemical Industries) following the manufacturer's protocol before embedding in paraffin. The percent metastatic area per tissue in the histological sections was calculated using Image J software. For immunohistochemical analysis, specimens were stained using rabbit anti-cadherin-17 IgG (sc-25628; Santa Cruz Biotech Inc., Santa Cruz, CA, USA) and HRP-conjugated goat anti-rabbit IgG (R&D).

Because the individuals were placed in elderly care facilities, the bodies were transferred from the facilities to our brain bank by hospital ambulance following death. At the time of autopsy, fresh brain tissue was dissected at the mid-sagittal line. The right cerebrum, cerebellum, and brainstem were immediately frozen by dry ice and stored at −80 °C for future studies. The left hemisphere of the brain was fixed in 20 % neutral-buffered formalin (Wako, Osaka, Japan) for neuropathological analysis.

Collected tissues were fixed using 10% neutral buffered formalin (Wako) or 4% paraformaldehyde (PFA, Wako) for 24 to 48 hr. After fixation, tissues were embedded in Tissue-Tek paraffin wax II 60 (Sakura Finetek Japan, Tokyo, Japan) and sectioned by a sliding microtome REM-700 (Yamato Kohki Industrial, Saitama Japan). Otherwise, tissues were embedded into NEG50 frozen section medium (Thermo Scientific), frozen, and sectioned by Cryostat CM3050 S (Leica Biosystems GmbH, Nussloch, Germany). The following dyes were used for staining the sections: hematoxylin (Wako)/eosin (Muto Pure Chemicals Co., LTD., Tokyo, Japan), Periodic acid-Schiff (Muto Pure Chemicals)/hematoxylin (Wako), or oil red O (Sigma–Aldrich)/hematoxylin (Vector). Some of the microscopic examinations were performed at BoZo Research Center Inc. Tokyo, Japan.

Wild type female BALB/c mice were purchased from Japan SLC, Inc. (Hamamatsu, Japan). 4T1 cells (ATCC, Manassas, VA, USA) were cultured in RPMI 1640 supplemented by 10% FBS, 2 mM L-glutamine, penicillin/streptomycin and sodium pyruvate. One million cells were grown to 50 to 80% confluence in T-75 tissue culture flasks. Cells were detached with 0.2% trypsin-EDTA, washed once with medium, three times with PBS and resuspended in PBS at 1 × 10⁶/mL. One hundred μL of cell suspension (1 × 10⁵ cells) were injected into the 3rd mammary pad of female mice. Mice were separated into 2 groups; 5 mice in the control group received i.p. injection of normal rat IgG (100 μg in 100 μL PBS) whereas 5 mice in the experiment group received anti-mouse GM-CSF IgG (100 μg in 100 μL PBS) twice a week, for 2 weeks. Blood was collected by heart puncture, and sera were isolated and stored at −80 °C until use. Tumors were harvested and a half was fixed in 10% neutral buffered formalin (Wako) and the other half was in RNAlater. Lungs were perfused with Bouin’s solution (Wako), fixed in the same solution, and then the number of tumor nodules was counted by eye. Tumor length and width were measured using a caliper and tumor volume was calculated using the following formula: Volume = (width)² × length/2.

To evaluate hypoxic effects of renal congestion, we conducted immunohistochemical analyses using the Hypoxyprobe^TM-1 kit (HP1-100, Hypoxyprobe, Inc., Burlington, MA) according to the manufacturer’s instructions. Under anesthesia, pimonidazole HCl (Hypoxyprobe^TM-1, 60 mg/kg bw, n = 3) was administered intraperitoneally immediately following. IVC ligation between the renal veins. Two hours later, both kidneys were removed and fixed with 10% neutral-buffered formalin (Wako Pure Chemical Industries Ltd., Osaka, Japan).

After the SPECT/CT imaging study, the mice were euthanized, and each tumour with brain was excised and frozen in Tissue-Tec optimal-cutting-temperature compound (Sakura Finetek). Frozen sections (20 µm thick) were fixed with 10% neutral buffered formalin (Wako), washed and dried. The dried sections were exposed to an imaging plate (Fuji Film, Tokyo, Japan), and the imaging plate was read using an FLA-7000 image plate reader (Fuji Film). After plate reading, the sections were stained with hematoxylin and eosin (HE). Tissue sections adjacent to those used for the autoradiographic study were immunostained with peroxidase-labelled anti-TF 1849 mAb. According to the manufacturer’s instructions, the peroxidase-labelled antibody was prepared using a peroxidase labelling kit (Dojindo, Kumamoto, Japan). The sections were fixed with 4% paraformaldehyde in PBS for 15 min at RT and endogenous peroxidase was blocked with 0.3% hydrogen peroxidase. After blocking with 5% skim milk in PBS overnight at 4 °C, the sections were incubated with peroxidase-labelled anti-TF 1849 mAb for 1 h at RT. The reaction was visualized by incubation with DAB (Dako), and hematoxylin was used for counter staining.