Ab170874

Proteins were extracted by SDS cell lysis buffer (P0013G; Beyotime) supplemented with protease inhibitor cocktail (87785; Thermo Scientific). Protein quantification was measured by the Pierce BCA protein assay kit (23225; Thermo Fisher Scientific). The protein bands were detected by conventional protocols for western blotting. Proteins were detected by using specific primary antibodies against secondary antibodies (Cell Signaling Technology). The following antibodies were used: monoclonal anti-GAPDH (ab181602,1:4000; abcam), monoclonal anti-TGFBI (ab170874, 1:1000; abcam), polyclonal anti-ZEB2 (abs131374, 1:1000; absin), monoclonal anti-VIM (cat#5741, 1:1000; Cell Signaling Technology), polyclonal anti-CTSL (ab200738, 1:1000; abcam), monoclonal anti-alpha tubulin (PTM-5442, 1:2000; ptmbiolabs), anti-rabbit IgG (7074, 1:2000; Cell Signaling Technology), and anti-mouse IgG (7076, 1:2000; Cell Signaling Technology).

Four-micrometer BPH sections and PCa tissue sections were cut and placed onto positively charged slides (MIC3040, Scientific Laboratory Supplies) and polyethylene naphthalate (PEN) membrane glass slides (LCM0522, Applied Biosystems). For every sample, four unstained sections were prepared. The immunostaining was performed using the primary antibodies DKK3 1:300 dilution (Ab187532 rabbit monoclonal, Abcam), ECM-1 1:200 dilution (11521-1-AP rabbit monoclonal, Proteintech), TGFBI 1:200 dilution (Ab170874 rabbit monoclonal, Abcam), and PCK-1 1:200 dilution (MA182041 mouse monoclonal, Invitrogen). EnVision+ System-HRP-labeled polymer anti-rabbit (K400311, DAKO) and anti-mouse (K400111, DAKO) were used as secondary antibodies.

Protein lysates were prepared in the presence of PIC (protease–inhibitor complex) and PMSF. Twenty microgram aliquots were separated on 8% sodium dodecyl sulphate polyacrylamide electrophoresis gels, and the proteins were transferred onto a polyvinylidene difluoride membrane (Merck Millipore). The membrane was incubated for 1 h in blocking buffer (tris-buffered saline containing 0.1% Tween (TBS-T), and 5% nonfat dry milk) followed by incubation overnight at 4 °C with the primary antibodies for DICER1 (CST, D38E7, 1:1000), TGFBI (Abcam, ab170874, 1:2000), FOXP1 (CST, 4402, 1:1000), FN1 (Santa Cruz, sc-9068, 1:200), CDH1 (CST, 3195s, 1:1000), and CDH2 (CST, 14215s, 1:1000). After washing with TBS-T, the blot was incubated with horseradish peroxidase (HRP)-conjugated secondary antibody or IRDye800-conjugated secondary antibody and the signals were visualized using an enhanced chemiluminescence system according to the manufacturer’s instructions (Kodak) or Odyssey (LI-COR). The uncropped and unprocessed scans of the most important blots are provided in the Source Data file.

According to the procedure described previously (27 (link)), formalin-fixed, paraffin-embedded liver tissues were used for immunohistochemistry (IHC) and immunofluorescence with the primary antibodies targeting at CD103, CD8, TGF-β, IL-15 (all anti-human, ab129202, ab17147, ab170874, ab55276, Abcam, Cambridge, United Kingdom), and FGL1 (anti-human, ab275091, Abcam; anti-mouse, 16000-1-AP, Proteintech, San Diego, CA, USA). Then information of antibodies could be found in the Supplementary table. For the quantification of IHC data in Cohort 1, five areas of each liver section were randomly selected and assessed by two independent observers. The expression of CD103⁺ were evaluated by calculating the number of each high-magnification field of view. The expression of FGL1 in cohort 1 was scored from 0 to 4 according to its staining density, which was averaged from five captured high-powered field. A tissue microarray (TMA) of 80 HCC tissues from cohort 2 was employed to assess the prognostic value of CD103 and FGL-1 according to the elaborated survival data. We quantified the expression of CD103 and FGL1 in each patient in TMA by measuring the number of positive cells per area (28 (link)). Confocal scans were performed using an LSM-710 laser scanning confocal microscope (Carl Zeiss, Germany).

For immunofluorescent multiple immunostaining, tissue samples were fixed in 10% buffered formalin and embedded in paraffin wax. In order to investigate cells expressing mesothelial markers in the adhesion tissues, triple fluorescent immunostaining with antibodies for PDPN (ab11936, Abcam), WT-1 (ab89901, Abcam) and α-SMA (ab124964, Abcam) was performed. To investigate proliferation of PDPN-positive cells in damaged ceca and adhesion tissues, double immunostaining was performed with antibodies against PDPN and Ki-67 (#12202 Cell signaling). In order to investigate whether neutrophils and/or mesothelial cells express TGF-β1, IL-6, and/or TNF-α, double immunostaining of TGF-β1, IL-6, and TNF-α in combination with Ly6G or α-SMA was performed. Antibody against for TGF-β1 (ab170874) was purchased from Abcam, and antibodies against for IL-6 (21865-1-AP) and TNF-α (17590-1-AP) were from Proteintech Group Inc. Fluorochrome labeling was performed with Opal 4-color fluorescent IHC kit (NEL820001KT, PerkinElmer) and viewed under a Zeiss LSM780 confocal microscope and documented using LSM780 software.