Fabp5

84 paraffin-embedded astrocytoma specimens with various grades were collected from the First Affiliated Hospital of Dalian Medical University. This study was approved by the hospital institution review board and informed consent was obtained from all patients. The expression patterns of CRABP-II and FABP5 in different grades of astrocytomas were profiled immunohistochemically, using paraffin sections of tissue microarrays constructed in duplicate with 84 astrocytomas and, where possible, noncancerous tumor surrounding tissues [38 (link)]. The antibodies used were rabbit anti-human CRABP II and FABP5 (Proteintech, Chicago, IL, USA) at dilutions of 1:100 and 1:80, respectively. Color reaction was developed using 3, 3′-diaminobenzidine tetrahydrochloride (DAB). The sections without the first antibody incubation were used as the background control. According to the labeling intensity, the staining results were evaluated by two researchers, and scored as negative (−) if no immunolabeling was observed in target cells, weakly positive (+) if the labeling was faint, moderately positive (++) if the labeling was stronger, and strongly positive (+++) if the labeling was distinctly stronger than (++) [39 (link)].

Western blotting was conducted using antibodies against CRABP2 (Proteintech, Chicago, IL, USA; 1:200), FABP5 (Proteintech, Chicago, IL, USA; 1:200), RAR-β (Bioss. Inc., Beijing, China; 1:150), PPAR-β/δ (Bioss. Inc., Beijing, China; 1:200), pro-caspases-3, and active-caspases-3 (Abcam Inc., Cambridge, UK; 1:500 and 1:500). The experiment was performed by the method described elsewhere [15 (link)]. Briefly, the sample proteins (20 µg/well) were separated by 10% SDS-PAGE electrophoresis and transferred to polyvinylidene difluoride membrane (Amersham, Buckin ghamshire, UK). The membrane was blocked in 5% skimmed milk (Sigma-Aldrich) Tris-buffered saline (TBS-T) (10 mM Tris–HCl, pH 8.0, 0.5% Tween 20 and 150 mM NaCl) at 4 °C overnight. After three washes with TBS-T, the membrane was incubated for 3 h with the primary antibody at room temperature, followed by 1 h incubation with horseradish peroxidase (HRP)-conjugated anti-rabbit IgG (Zymed Lab Inc., San Francisco, CA, USA). The enhanced chemiluminescence system (Roche, Penzberg, Germany) was used to detect the bound antibody. The labeling signal was removed with a stripping buffer (62.5 mM Tris–HCl, pH 6.7, 100 mM 2-mercaptoethanol, 2% sodium dodecyl sulfate (SDS), and the membrane was reprobed with another antibody until all the parameters were examined.

Immunohistochemistry was performed as described previously (Ryu et al., 2018 (link)). Paraffin-embedded breast tumor tissues were sectioned by the pathology laboratory of the Fourth Hospital of Hebei Medical University (Shijiazhuang, Hebei, China) at a thickness of 5 microns. Free-floating slides were cultured in a PBS solution containing 3% H₂O₂ (v/v), rinsed in PBS three times, and blocked for 30 min at 37°C with 5% goat serum. The slides were incubated with FABP5(1:200, proteintech, USA) and p-CaMKII (1:200, ABclonal, China) overnight at 4°C. After washing, the slides were cultured for 30 min at 37°C with secondary anti-rabbit IgG (proteintech, USA), and were subsequently scanned via a pathology section scanner (Pannoramic SCAN, 3D HISTECH, Hungary) to observe the expression of FABP5 and p-CaMKII.

Paraffin-embedded ovarian tissues from PCOS and normal mice were obtained based on Li’s work [25 (link)]. Tissue sections were dewaxed and treated with freshly prepared PBS containing 0.3% hydrogen peroxide for 10 min to block endogenous peroxidase activity. Antigen retrieval was conducted by autoclaving the samples at 121 °C for 15 min in the presence of EDTA (pH = 9.0), followed by incubation in blocking solution to block endogenous biotin for 30 min. The sections were washed with TBS and then stained overnight with the primary antibody FABP5 (1:500; Proteintech, Chicago, USA) at 4 °C. Subsequently, the sections were rinsed with PBS and incubated with an HRP-conjugated goat anti-rabbit secondary antibody (rabbit ABC detection kit, ZSBio, Beijing, China) at 37 °C for 30 min. Next, the sections were stained with 3,3’-diaminobenzidine (DAB) and counterstained with hematoxylin. Control sections were prepared concurrently with the experimental sections and treated with nonspecific rabbit IgG. Nonspecific staining was not detected in the controls.