Oct compound

Tumour cells were isolated using LMD (Leica LMD6000, Leica Microsystem). Briefly, frozen tumour tissue was embedded in optimal cutting temperature (OCT) compound (Leica Microsystems, Wetzlar, Germany), cut into 20 µm thick sections using Leica CM1850 UV Cryostat (Leica Microsystems) at − 20 °C and mounted onto PET-membrane steel frame slides (Leica Microsystems). The sectioned tissues were fixed with 70% ethanol for 30 s, and stained in Harris’ haematoxylin (VWR, England) for 30 s then rinsed in water and air dried. All staining procedures were carried out on ice with all solutions supplemented with fresh 1 mM phenylmethanesulfonyl fluoride (Sigma, St Louis, MO, USA). The estimated total protein yield from one thousand cells was approximately one µg protein.

The isolated bone of a hind limb was fixed in 70% ethanol for 48 h, decalcified in 10% EDTA for 2 weeks, processed, and embedded in paraffin. Sectioned bones (10 μm thickness) were then stained with hematoxylin–eosin. To detect bone metastasis of mKO2-expressing cancer cells, isolated bones were embedded in OCT compound (Leica Microsystems, Wetzlar, Germany), and cryosections of the embedded bones were prepared by using CM3050 Cryostat (Leica BIOSYSTEMS), stained with Hoechst and observed under a confocal fluorescence microscope (LSM700, Carl Zeiss, Oberkochen, Germany). To quantitatively evaluate the formation of bone metastatic lesions, multiple sections were prepared from eight bones dissected from CA and IC-injected mice and metastatic lesions in each section were counted. The size of bone metastasis was quantifies by measuring area with mKO2 fluorescence with ImageJ software.

At appropriate time points, mice were sacrificed and lungs were transcardially
perfused with 0.9% NaCl prior to fixation in 4% paraformaldehyde (PFA) in
0.1 M phosphate buffer at 4 °C overnight.
After washing in ddH₂O the lungs were first transferred into a 15%
sucrose solution in 0.1 M phosphate buffer (w/v) for 4 h
and thereafter in a 30% sucrose solution in 0.1 M phosphate buffer
(w/v) overnight (4 °C). For cryosection, lungs were
embedded within Tissue-Tek O.C.T. compound, solidified on dry ice and cut to
15 μm thickness using a cryotome (Leica Microsystems,
Germany). The sections were mounted onto gelatine-coated slides and dried at
room temperature overnight in the dark. The slides were washed twice in
phosphate buffer, DAPI (Invitrogen) stained at an end concentration of
300 nM in 0.1 M phosphate buffer for 7 min
and again washed 3 times in 0.1 M phosphate buffer. Dried slides
were embedded within IMMU-Mount™ (ThermoShandon), coverslipped and
stored in the dark at 4 °C until further use.

We obtained de-identified TNBC clinical specimens from BDDR at RPCCC with Institutional Review Board approval (RPCCC, BDR # 074516). ST6GAL1 expression was evaluated by immunohistochemistry in the TNBC tissue section of one individual, as previously described [18 ]. The tissue specimen was embedded in an OCT compound (Leica Microsystems, Wetzlar, Germany), and cryosections of the embedded tissues were prepared. Slides were blocked in 5% BSA for 1 h, incubated overnight with anti-ST6GAL1 antibody, then with anti-goat-HRP secondary (R&D Biosystems) for 1 h. Tissues were then immersed in Impact DAB stain (Vector Labs) for 120 s and rinsed in water for 3 min. Images were acquired under 200× and 630× (insert) magnifications with a Nikon Eclipse E600 Optical microscope. Spot RT3 camera and Spot Software were used to capture and analyze the mages. Dark brown (red arrow) and light brown (green arrow) of ST6GAL1 stainings are indicated as a heterogeneous expression pattern of ST6GAL1 protein.

For in vitro immunofluorescence analysis, Caco-2 cells were seeded at a density of 1 × 10⁵ cells per mL on an eight-well chamber (ibidi, GmbH, Gräfelfing, Germany) for 3 weeks. Cells were pretreated with 25 and 50 μM sinapic acid for 1 h and then treated with the stimulus. For in vivo immunofluorescence analysis, colon tissues were fixed using an OCT compound (Leica Microsystems, Seoul, Republic of Korea) and cryosectioned into 10 μM sections. Cells and sectioned colon tissues were fixed with 4% formaldehyde for 15 min and washed three times with PBS. Cells and sections were permeabilized with ice-cold 100% methanol and then blocked and incubated overnight at 4°C with the primary antibodies occludin (1:100) and ZO-1 (1:200). After washing, the cells and sections were incubated with a goat anti-rabbit immunoglobulin G H&L conjugated to the Alexa Fluor^® 488 and 647 secondary antibodies (Abcam, Cambridge, United Kingdom) for 1 h. Subsequently the cell nucleus was stained with VECTASHIELD^® Antifade Mounting Medium with DAPI (Vector Laboratories Inc., Burlingame, United States).

For cryoprotection, brains were gradually immersed in 10, 20, and 30% sucrose in 24 h intervals and embedded in OCT compound (Leica Microsystems, Bensheim, Germany) with liquid nitrogen. The brains were cut into frozen coronal sections (30 μm) using a Leica CM3050 cryostat and then stored in a free-floating buffer. For immunohistological analysis, the sections were blocked in 5% normal chicken serum (containing 0.3% Triton X-100) for 1 h. After washing, the sections were incubated with primary antibodies against ionized calcium-binding adaptor molecule 1 (Iba-1, 1:200, 019-19741, Wako) or 5-hydroxytryptamine (5-HT, 1:200, ab66047, Abcam) overnight at 4°C. Subsequently, the sections were incubated with goat anti-rabbit IgG HRP (1:400, ab6722, Abcam) or Alexa Fluor^® 488 donkey anti-goat IgG H&L (1:400, ab150129, Abcam) secondary antibodies for 2 h at RT. To analyze the Iba-1-positive cells, the sections were incubated with an avidin-biotin-peroxidase complex (VECTASTAIN ABC kit, Vector Laboratories) for 2 h. The peroxidase activity was visualized using a stable diaminobenzidine solution. To analyze the 5-HT-positive signal, the sections were exposed to DAPI (1:1,000, D9542, Sigma) to stain cell nuclei. Immunoreactivity was observed under an Axio-Phot microscope (Carl Zeiss, Germany). The intensity was quantified using ImageJ 1.46 software (NIH, Bethesda, MD, USA).

Isolated whole bladders were pre-fixed in 4% paraformaldehyde overnight and cryoprotected in 30% sucrose. The samples were embedded in OCT compound (Leica Microsystems, Germany), cryosectioned into 10 m thick slices, and mounted on microscope slides. After permeabilization in a solution containing 0.5% triton X-100 and 6% bovine serum albumin for 1 hour, the sections were incubated overnight at 4 °C with primary antibody [Anti- -smooth muscle actin (#ab21027, Abcam), 1:200]. The sections were then washed three times with 1X PBS and incubated with the secondary antibody [Alexa Fluor 594 Donkey Anti-Rabbit IgG (#A21207, Invitrogen), 1:1000] for 1 hour at room temperature. The sections were subsequently washed three times with PBS and stained with DAPI (#R37606, Molecular Probes) to visualize the nuclei. Finally, the sections were rinsed with PBS three times and mounted with Clearmount (#008010, Invitrogen, UK). Confocal fluorescence images were acquired using a ZEISS scanning laser microscope with a 20X or 40X oil immersion objective lens (CLSM780, ZEISS, Germany).