Vector dab kit

Mouse xenograft tumors were collected at the end of treatment and immediately fixed in 10% neutral buffered formalin. Samples were subjected to embedding in paraffin and sectioning at 5 µm. The resulting sections were used for haematoxylin and eosin (H&E) and immunohistochemistry staining. The following primary antibodies were used: DHODH (1:100; Proteintech), 4HNE (5 µg/ml; Jaica), and Ki67(1:100; Proteintech). A goat anti-rabbit IgG and a goat anti-mouse IgG were used as secondary antibodies. Immunohistochemical staining was performed using a Vector DAB kit.

Kidneys were regularly harvested for SLC2A1 and GPX4 immunohistochemistry (IHC). Following overnight fixation in 4% paraformaldehyde, the tissues were embedded in paraffin and sectioned at a thickness of 4 μm. After subjecting the sections to antigen retrieval in a solution containing 10 mM sodium citrate, 0.05% Tween-20 at a pH of 6.0, they were heated at 95–100°C. To block endogenous peroxidase activity, the slides were treated with 3% H₂O₂. Additionally, 2.5% normal horse serum was applied to minimize nonspecific binding. The slides were subsequently incubated at a temperature of 4°C overnight with rabbit anti-SLC2A1 (21829-1-AP, 1:200, Proteintech), anti-GPX4 (HuaAn, Hangzhou, China) and then treated with ImmPRESS HRP polymer horse-anti-rabbit secondary antibody at room temperature for 1 hour. Negative controls were established by substituting the primary antibody with antibody diluent. The signals were visualized using the Vector^® DAB kit and counterstained with hematoxylin following the washing steps. To quantify the staining, a total of 20 to 30 fields (400× magnification) were randomly chosen from each section. The percentage of positive stained area was then analyzed utilizing ImageJ software.

The paraffin sections were prepared as described above. The sections were de-waxed in xylenes, re-hydrated, and boiled for 10 min in 10 mM sodium citrate buffer (pH 6.0) using a microwave. Most primary antibodies, such as anti-RHAU, were treated with the sodium citrate, whereas anti-BG4 with trypsion pretreatment (25 mg Trypsin, 314 mg CaCl₂.2 H₂O in 100 ml 50 mM Tris, PH7.8) incubated for 20 min at 37 °C to prevent G4 DNA forming at an artificial state. After treating with 0.3% hydrogen peroxide for 10 min, these paraffin sections were incubated with blocking solution (TBST/5% normal goat serum). Then, we washed away the blocking solution, added primary antibodies, and washed the samples three times in Tris-borate-EDTA buffer (shorted for TBS). Next, biotinylated second antibodies were added to the samples, and ABC reagent (Vector ABC kit, PK-6100) was prepared conforming to the manufacturer's instructions. Then, DAB reagent (Vector DAB kit, SK-4100) was used to stain the slides. Eventually, the slides were stained with hematoxylin following the manufacturer's instructions.

One uterine horn per mouse was fixed overnight in 10% neutral buffered formalin, processed in increasing concentrations of ethanol, and paraffin embedded. Tissues sections (5μm) were processed for histological analysis. Sections were stained with Hematoxylin and Eosin and analyzed by two pathologists blinded to the experimental exposures. For immunohistochemistry, sections were incubated with antibody specific to phospho-histone H3 (pHH3), alpha smooth muscle actin (αSMA) and cytokeratin 8 (CK8) (S1 Table), then with goat HRP-conjugated secondary antibody (Santa Cruz Biotechnology, 1:1000). Slides were developed for 5 minutes with a Vector DAB Kit (Vector Laboratories) per manufacturer's protocol, counterstained in CAT Hematoxylin. Representative images were taken on a Nikon Eclipse E800 upright microscope with an Olympus DP71 camera and manufacturer's software. We used primary antibody against pHH3 for immunofluorescence (S1 Table) followed by goat Alexa Fluor 488—conjugated secondary antibody (Molecular Probes, 1:1000). Sections were counterstained with DAPI imaged by confocal microscopy (Leica TCS SPE/DMI4000B; Leica Application Suite software).

Immunohistochemical staining was performed using 5-μm-thick sections of 4% paraformaldehyde-fixed paraffin-embedded tissue samples. The following primary antibodies were used: anti-AGPAT3 antibody (1:200; DF3642; Affinity; RRID: AB_2836014), anti-4HNE monoclonal antibody (5 µg/ml; Jaica), and anti-AGPS antibody (1:700; ab236621; Abcam; RRID: AB_2921211). Goat anti-rabbit IgG and goat anti-mouse IgG were used as secondary antibodies. Immunohistochemical staining was performed using a Vector DAB kit. Notably, immunohistochemical staining was performed to examine the expression profiles of AGPS and AGPAT3 in gastric specimens as described previously; the expression profiles were evaluated and scored by two pathologists at Nanfang Hospital for the staining intensity (scale = 0–3) and staining frequency (scale = 0–4). For statistical analysis, the expression levels of the AGPS and AGPAT3 proteins were represented by an expression score ranging from 0 to 12 and calculated using the formula intensity×frequency.

On the 28th day after SCI, bilateral and stereotaxical injections of biotinylated-dextran amine (BDA; MW 10,000, 10%, 1 μl/site; Molecular Probes, Eugene, OR) were made into the intermediate gray matter of the T7-8 cord segments at distances of 3 – 6 mm rostral to the contusion site (for BDA, one injection/site/mm longitudinal distances) according to previously published work (Deng et al., 2013 (link)).
BDA staining was performed according to earlier reports (Fouad et al., 2001 (link)). Slides were dried in an incubator at 38°C for 1 hr and washed twice for 10 min in 50 mM Tris-buffered saline (TBS), pH 7.4, followed by two 45 min washes with TBS containing 0.5% Triton X-100. Afterwards, the slides were incubated overnight with an avidin–biotin–peroxidase complex in TBS with Triton (ABC Elite, Vector Laboratories, Burlingame, CA) according to the manufacturer’s protocol. Subsequently, a DAB reaction was performed using the Vector DAB kit (SK4100, Vector Laboratories). The reaction was monitored and stopped by extensive washing in water.

Two months after the therapeutic intervention, animals were euthanized with an overdose of pentobarbital sodium (80 mg/kg) and an intracardiac perfusion with 4% paraformaldehyde was performed. The affected portions of the SC were fixed overnight and then transferred to 30% sucrose for cryoprotection. Samples were embedded in Tissue-Tek (Miles Elkhart, IN, USA), and longitudinal frozen sections (40 μm thick) were performed. Immunohistochemical staining was carried out in order to count the amount of TH+ and 5-TH+ fibers. Tissues were incubated in 0.03% hydrogen peroxide to quench endogenous peroxidase activity. Subsequently, the tissue was incubated overnight with the following primary antibodies: monoclonal goat antibody against TH (1:2000; Chemicon), or polyclonal rabbit antibody against 5-HT (1:2000; Sigma- Aldrich). Following rinsing with PBS, samples were incubated for at least 2 h with donkey IgG anti-goat IgG (1:500; Chemicon) and Sheep IgG anti rabbit IgG (1:500; Abcam) secondary biotinylated antibodies. To visualize positive fibers, samples were incubated 5 min with Vector DAB kit (Vector laboratories, CA, USA). Then, samples were evaluated and analyzed by a blinded observer that counted individual fibers using a 20X objective (Olympus DP72, Japan). The number of regenerating axons 1 mm caudal to the lesion was assessed.