Ab32351

Paraffin sections were deparaffinized and hydrated. After blocking the endogenous activity of peroxidase using 10% hydrogen peroxide, the sections were blocked for non-specific reactions and incubated with primary rabbit polyclonal antibodies against Cytochrome c (SAB4502234; 1:50–1:100 dilutions; Sigma-Aldrich, St Louis, MO, USA), rabbit monoclonal antibodies against caspase-3 (ab32351; 1/25–1/100 dilutions; Abcam, Cambridge, UK), mouse monoclonal [C2C12] against NCX1 (ab2869; 5 µg/mL concentration; heat-mediated antigen retrieval performed with citrate buffer of pH 6 before commencing with IHC staining protocol; Abcam, Cambridge, UK), and rabbit polyclonal to SERCA2 ATPase (ab3625; 1 µg/mL concentration; heat-mediated antigen retrieval performed before commencing IHC staining protocol; Abcam, Cambridge, UK). Then, after washing with phosphate buffer, a biotinylated goat anti-rabbit secondary antibody was applied. For localization of the immune reaction, the slides were incubated with labeled avidin-biotin peroxidase, which binds to the biotin on the secondary antibody. Diaminobenzedine was used as chromogen for visualization of the site of antibody binding, which is converted into a brown precipitate by peroxidase [96 (link)].

Western blot was performed as described previously [27 (link)]. HUVECs were inoculated into 60 mmol/L culture dishes. When the adherent growth reached 80%, different treatment factors were given. The culture medium was discarded, washed twice with precooled PBS, and added 80 μL lysis solution to each dish and cook at 4°C for 30 min. Centrifugation at 12,000 r/min for 15 min, took the supernatant, and determined the protein concentration by BCA method. The total protein was separated by SDS-PAGE and transferred to PVDF membrane (Beijing pulley gene). Sealed with 5% skimmed milk powder for 90 min, and added various primary antibody SFRP5 (ab230425, 1:1000, Abcam), Wnt5a (ab282153, 1:500), p-JNK1/2/3 (ab124956, 1:1000), caspase3 (ab32351, 1:5000), Bax (ab32503, 1:1000), Bcl-2 (ab182858, 1:2000) and TGF-β1 (ab215715, 1:1000), 4°C overnight, then washed with Tris Buffered Saline with Tween (TBST) for 3 times (10 min each time), added secondary antibody (1:1000), incubated at room temperature for 60 min, and then washed with tbst for 3 times (10 min each time). The PVDF film was colored by ECL solution, exposed to darkroom, and analyzed by gel imaging system.

U937-derived macrophages or BMDM were detached using trypsin-EDTA solution (U937) or 1× PBS containing 1 mM EDTA (BMDM), washed twice in ice-cold 1× PBS, and lysed for 20 min in ice-cold lysis buffer (50 mM HEPES [pH 7.4], 5 mM 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate [CHAPS], 5 mM dithiothreitol [DTT]). During this procedure, cells were kept on ice. Cell lysates were centrifuged for 10 min at 18,000 × g and 4°C. Supernatants were mixed with sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE) loading buffer and boiled at 95°C for 10 min. Proteins were separated on a 12% SDS-PAGE gel and transferred onto polyvinylidene difluoride (PVDF) membranes for immunoblot analysis with the following rabbit primary antibodies: anti-caspase-3 (anti-CASP3; for U937, antibody ab32351, and for BMDM, antibody ab13847, both from Abcam) and anti-GAPDH (loading control, PA1-987; Thermo Fisher; GAPDH, glyceraldehyde-3-phosphate dehydrogenase). Immunoreactive signals were revealed with a secondary antibody conjugated to horseradish peroxidase (Cell Signaling, Danvers, MA, USA); horseradish peroxidase activity was detected with enhanced chemiluminescent (ECL) substrate.

Immunohistochemical staining of caspase-3 (CAT number: ab32351, Abcam company 152 Grove Street Waltham, M02453, USA) and synaptophysin (CAT number: ab32127, Abcam company 152 Grove Street Waltham, M02453, USA) were performed by using rabbit monoclonal antibodies. For Bcl-2 (CAT number: ab59348, Abcam company 152 Grove Street Waltham, M02453, USA), it took place by using rabbit polyclonal antibodies. They were received in a single vial containing 1 ml of antibody. Anti-caspase-3, anti-synaptophysin, and anti-Bcl-2 were used in diluted quantities 1:50, 1:400, and 1:100 respectively. Sections were cut at 5 µm and stained by an automated LINK 48 immunostainer (Dako, Agilent Technologies Inc, Santa Clara, USA). For heat retrieval, citrate buffer was used by immunostainer. Slides were stained automatically by primary diluted antibodies. Positive control for the reaction was performed using specific paraffin-embedded sections of the normal human tonsil, human pancreas, and follicular adenoma for caspase-3, synaptophysin, and Bcl-2, respectively. Negative controls were made by substituting the primary antibodies with non-immune serum.
Assessment of histopathology and immunohistochemical markers (caspase-3, synaptophysin and Bcl-2) was done in different areas of the hippocampus including CA1,CA2 and CA3.

Each group's fresh ovarian tissue was removed, and the P1 generation of TICs was cultivated until it reached 90% confluence. Nuclear protein extraction kit (Solarbio, Beijing, China)was used to extract nuclear and cytoplasmic protein from TICs as well as total protein using RIPA pyrolysis. SDS-PAGE was used to separate the proteins, and they were then moved to the polyvinylidene difluoride membrane (PVDF). The membranes were incubated with the primary antibodies Cyp17a1 (1:4000 dilution; ab125022; Abcam), NR4A1 (1:500 dilution; sc-166166; Santa Cruz), Phospho-NR4A1 (1:1000 dilution; Ser351; Cell signaling technology), Bcl-2 (1:1000 dilution; Abclonal), Bax (1:1000 dilution; Abclonal), Caspase-9(1:1000 dilution; Abclonal), caspase-3(1:500 dilution; ab32351; Abcam) and cytc(1:500 dilution; Cell signaling technology) at 4℃ overnight, following being washed three times with TBS added with Tween 20(TBST). The second antibody was administered at room temperature for one hour and was HRP-conjugated affinipure goat anti-rabbit IgG (H + L) (1:40,000 dilution; 10,285–1-AP; Proteintech). Utilizing the Tanon 5200 analytical equipment (Tanon, Shanghai, China), chemiluminescence was measured following reaction with the Ultra-sensitive chemiluminescence kit (Sparkjade, Qingdao, Shandong).