Ab27969

Specimens (including 5 normal human brain tissue samples and 5 GBM tissue samples) and cells were performed using antibodies against NOX4 (ab13303, 1 : 200, Abcam, USA), TGF-β1 (ab27969, 1 : 100, Abcam, USA), Vimentin (ab92547, 1 : 200, Abcam, USA), and HIF-1α (20960-1-AP, 1 : 50, Proteintech, China). Fluorescent secondary antibodies (anti-rabbit IgG, SA00013-2, 1 : 500 and anti-mouse IgG, SA00013-3, 1 : 500, Proteintech, China) were used to detect the primary antibodies.

A Western Blot (WB) investigation was carried out to evaluate TGF-β1 protein content in samples’ CM. In detail, equal volume amount (30 µL) of CM collected from CTR, and cells seeded on PLGA fleeces and 3D scaffolds, were processed for WB as previously described [38 (link)]. The total protein amount in CM has been normalized on a Ponceau S stain, according to Sander et al. [39 (link)]. TGF-β1 expression was detected using a specific primary antibody (Abcam, ab27969, Milan, Italy) diluted (1:250) in tris-buffered saline (TBS) solution, and incubated overnight at 4 °C. Finally, the WB membranes were incubated for 1 h at RT with an anti-mouse secondary antibody conjugated to horseradish peroxidase (HRP) (Santa Cruz, sc-516102, Heidelberg, Germany) diluted in TBS (1:10,000). The ECL substrate was used to visualize the target protein (LiteAblot PLUS, Euroclone, Milan, Italy) and Azure’s 400 detected the chemiluminescent signal (Azure Biosystems, c400, Sierra Ct, Dublin, CA, USA). Image J blot analyzer software was employed for the densitometric analysis (ImageJ 1.53k, NIH, Bethesda, MD, USA).

IHC and IF experiments were carried out as described previously (He et al., 2014; Miyamoto et al., 2012). Tumor sections were placed on slides and were incubated with the following primary antibodies, mouse monoclonal to ERβ (14C8, Abcam; dilution 1 : 50), rabbit anti‐TGFβ1 polyclonal antibody (ab27969, Abcam; dilution 1 : 100), N‐cadherin rabbit mAB (D4R1H, Cell Signaling Technology, Beverly, MA, USA; dilution 1 : 200) and anti‐luciferase (Santa Cruz Biotechnology) in 3% BSA in PBS overnight at 4 °C, followed by appropriate secondary antibodies. The stained slides were mounted and visualized by a bright‐field microscope or a fluorescence microscope.

The cells were washed with 1 × PBS and fixed with 4% paraformaldehyde for 30 min at 25 °C. Next, 0.1% Triton X-100 was added and incubated at 25 °C for 10 min. Thereafter, we added PBS containing 2% bovine serum albumin (BSA) for 30 min for blocking. Anti-TRP1 antibody (ab235447; Abcam) was diluted at a 1/200 ratio in PBS containing 2% BSA for 2 h at 25 °C. Anti-CD68 antibody (ab955; Abcam), anti-TGF-β antibody (ab27969; Abcam), and anti-IL-6 antibody (ab9324; Abcam) were diluted in a 1/200 ratio in PBS containing 2% BSA overnight at 4 °C. Alexa Fluor^®594-conjugated goat anti-rabbit IgG (H + L) antibody (A11037, Molecular Probes, Eugene, OR, USA) and Alexa Fluor^®488-conjugated goat anti-mouse IgG (H + L) antibody (A11029, Molecular Probes) were diluted in PBS containing 2% BSA and incubated for 1 h at 25 °C. Finally, 4,6-diamidino-2-phenylindole (DAPI; 10236276001; Roche) was applied at 25 °C for 10 min. The cells were washed with PBS and images were obtained using an upright fluorescence microscope (Axio Imager.M2; Carl Zeiss, Oberkochen, Germany) for iMel and confocal laser scanning microscope (LSM800 w/Airyscan; Carl Zeiss) for macrophages.

The mouse-anti-human monoclonal antibody R6G9 (IgG_2a), which is directed against the extracellular domain of human integrin subunit β6, was obtained from Chemicon International (Temecula, CA, U.S.A.). The following monoclonal antibodies were obtained from Abcam (Cambridge, MA, U.S.A.): ab5694 and ab28244, which target the CAF markers α-SMA and FAP, respectively: ab27969, which targets transforming growth factor β (TGF-β); and ab9797, ab9695, ab16828, and ab6672, which target the four cytokines stromal cell-derived factor-1 (SDF-1), epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), and interleukin 6 (IL-6). EP1186Y and EP1254 monoclonal antibodies, which target matrix metalloproteinase (MMP) 3 (MMP-3) and MMP-9, respectively, were also purchased from Abcam. Reagents for SDS/PAGE and molecular weight markers were obtained from Bio-Rad Laboratories (Hercules, CA, U.S.A.). The C–X–C chemokine receptor type 4 axis (CXCR4) antagonist AMD3100 was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, U.S.A.).

After lysing in RIPA lysis buffer, the collected total protein was separated on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) (10%), shifted onto polyvinylidene fluoride membranes, and then blocked by 5% nonfat milk. The membranes were probed all night at 4 °C with the primary antibodies against loading control GAPDH (ab8245, 1/1000; Abcam, Cambridge, MA, USA) and p65 (ab16502, 1/1000; Abcam), Histone H3 (ab1791, 1/1000; Abcam), IKKα (ab32041, 1/10,000; Abcam), IKKβ (ab124957, 1/1000; Abcam), NEMO (ab178872, 1/5000; Abcam), p-IKBα(Ser36) (ab133462, 1/10,000; Abcam), p-IKBβ(Ser23) (Cat# 4921 S, 1/1000; Cell Signaling Technology, Danvers, MA, USA), CD9 (ab92726, 1/2000; Abcam), CD63 (ab134045, 1/1000; Abcam), CD81 (ab109201, 1/1000; Abcam), HSP70 (ab2787, 1/1000; Abcam), E-cadherin (ab76055, 1/1000; Abcam), α-SMA (Cat# 19245S, 1/1000; Cell Signaling Technology), TGF-β1 (ab27969, 1/2000; Abcam), Collagen type I (ab34710, 1/1000; Abcam), Collagen type III (ab7778, 1/5000; Abcam), Usp5 (ab154170, 1/1000; Abcam). Next, membranes were washed in TBS-T, and then incubated for 2 h with horseradish peroxidase-labeled secondary antibodies at room temperature. Electrochemiluminescence luminous liquid was employed for analyzing protein bands as instructed by supplier (Pierce, Rockford, IL, USA). Results were visualized after developing in the dark.