Normal swine serum

The use of fresh liver tumor specimens was approved by the research ethic committees at the Catholic University (South Korea). Informed consent was obtained from all patients. Samples were fixed with 4% paraformaldehyde for fluorescent staining. Samples were permeabilized with 0.4 M glycine and 0.3% Triton X-100, and nonspecific binding was blocked with 2% normal swine serum (DAKO, Glostrup, Denmark). Staining was performed as described previously [57 (link)], using the primary anti-ALDH1 (Abcam, Cambridge, MA, USA, ab52492), CD133 (Biorbyt, Cambridge, UK, orb114000), Wnt1 (Abcam, Cambridge, MA, USA, ab15251), β-catenin (Cell Signaling Technology, Beverly, MA, USA, #9562), and PCNA (Vector Laboratories, Burlingame, CA, USA, VP-RM04) antibody. Samples were examined by fluorescence microscopy (Zeiss LSM 510 Meta). The calculation of expression was based on green fluorescence area and density divided by cell number, as determined from the number of DAPI-stained nuclei, in three randomly selected fields for each sample from a total of three independent experiments.

Human tumor samples were obtained from colorectal cancer patients with informed consent and all of the experiments were approved by the Institutional Review Board of Gil Hospital (GCIRB-2013-66). Samples were fixed with 4% paraformaldehyde for fluorescent staining. Samples were permeabilized with 0.3 M glycine and 0.3% Triton X-100 and nonspecific binding was blocked with 2% normal swine serum (DAKO, Glostrup, Denmark). Staining was performed as described previously [38 (link)], using the primary SERPINB2 (Abcam, MA, USA, ab47742), CD44 (NOVUS, NBP1-48386), CD133 (NOVUS, NB120-16518) ALDH (Abcam, ab23375) antibodies. Alexa Fluor 488-conjugated rabbit IgG (Molecular Probes, Eugene, OR) was used to visualize SERPINB2. Samples were examined by fluorescence microscopy (Zeiss LSM 510 Meta, Oberkochen, Germany). The calculation of SERPINB2 expression was based on green fluorescence area and density divided by cell number, as determined from the number of DAPI-stained nuclei, in three randomly selected fields for each specimen from a total of three independent experiments. For quantitation, an arbitrary threshold was set to distinguish specific from background staining and this same threshold setting was applied to all the samples analyzed.

Cultures of ESC were fixed with 4% paraformaldehyde, permeabilized with 0.1% Triton X-100 in PBS and blocked with 10% normal swine serum (Dako). Incubation of cell or tissue samples with primary antibodies was performed at 4°C overnight, followed by incubation with secondary antibodies for 30 mins at 37°C after three thorough washes with PBS. Subsequently, the cells were counterstained with DAPI (1:1000 in PBS) for 3 mins at room temperature and mounted with fluorescent mounting media (Dako) before image acquisition using an Axio Imager. M2 microscope and AxioVision Digital Imaging System (Carl Zeiss Ltd.). Primary antibodies used were Calponin, SM-22α (both from Abcam), SM-αA (Sigma Aldrich), HSP47 (Abcam) and DAPI (Sigma Aldrich). The appropriate fluorescent-conjugated (Alexa 488 and Alexa 546) IgG antibodies were used as secondary antibodies (Invitrogen).

Deparaffinised EAT sections were rehydrated and antigen was retrieved by autoclaving in sodium citrate buffer 0.01 M pH 6 for 5 minutes at 120 °C. After rinsing in PBS 1X, endogenous peroxidase activity was quenched in 0.3% H₂O₂ in PBS for 20 minutes. To block unspecific binding, sections were incubated with normal swine serum (Dako Cytomation) and then with the following primary antibodies: mouse monoclonal anti-human ILRL1 (diluted 1:400 in PBS, Proteintech) and mouse monoclonal anti-human IL-33 (diluted 1:200 in PBS, Proteintech), both overnight. Sections were rinsed in PBS and processed for amplification of the immune signal with anti-mouse HRP-polymer complex (MACH 1 Universal HRP-Polymer detection, Biocare Medical). Biocare’s Betazoid DAB was used to develop color. Sections were counterstained with Mayer’s hematoxylin and mounted with Mowiol 4–88.
Immunohistochemical reactions were observed with a Nikon Eclipse 80i microscope and images were acquired with a digital camera and the image acquisition software.

Deparaffinised EAT sections were rehydrated and antigen retrieval was performed by autoclaving in sodium citrate buffer 0.01 M pH 6 for 5 min at 120 °C. After rinsing in PBS 1X, quenching of endogenous peroxidases activity was performed in 0.3% H₂O₂ in PBS for 20 min. To block unspecific binding, sections were incubated with normal swine serum (Dako Cytomation) and then with the following primary antibody: mouse monoclonal anti-human EPAC2 (diluted 1:400 in PBS, Cell Signaling) overnight and overnight. Sections were then rinsed in PBS and processed for the amplification of immune signal using anti-mouse HRP-polymer complex (MACH 1 Universal HRP-Polymer detection, Biocare Medical, Concord, CA, USA). BIOCARE’s Betazoid DAB was used for color development Sections were counterstained with Mayer’s hematoxylin and mounted with Mowiol 4–88.
Immunohistochemical reactions were observed with a Nikon Eclipse 80i microscope and images acquired by the digital camera and the image acquisition software.