Brdu in situ detection kit

Formalin-fixed paraffin-embedded sections were deparaffinised, rehydrated, antigen-retrieved for 15 min in sodium citrate at subboiling temperatures and peroxidase-blocked. The primary antibody was incubated over night at 4 °C and the secondary biotinylated antibody mediated horseradish peroxidase (HRP)-driven 3,3′-diaminobenzidine (DAB, DAKO, K3468) turnover, which resulted n brown labeling of immunoreactive cells. Stained sections were analyzed with a light microscope (Zeiss, Germany) and captured with a Zeiss AxioCam. TUNEL labeling was performed according to the manufacturer’s instructions (Roche, 11684817910). TUNEL-positive cells were quantified in 50 consecutive crypts and depicted as TUNEL⁺ cells of total IECs. Proliferation of IECs was investigated by intraperitoneal injection of 5-bromodeoxyuridine (BrdU, BD Pharmigen, 550891) and detected after 24 h using a BrdU in situ detection kit (BD Pharmigen, 550803). BrdU⁺ cells of total IEC along the villus-crypt axis were analyzed. PAS reaction was performed according to a standard protocol and PAS⁺ cells were counted in 50 crypt/villus axes.
The following antibodies were used for immunohistochemistry: anti-GPX4 (1:400, Abcam, ab125066), anti-4HNE (1:400, Abcam, ab46545) and anti-MPO (1:200, Dako, IS511) were employed with a secondary biotinylated antibody (Vector, MP-7401).

BrdU labeling was performed using BrdU In-Situ Detection Kit (BD Biosciences, 550803) according to the manufacturer’s instructions. Briefly, the mice were i.p. injected with 1 mg of BrdU and the tissues were collected from the injected mice at 24 hr post injection, followed by paraffin embedding and sectioning. After being deparaffinized and antigen-retrieved, the section was stained using biotinylated anti-BrdU and Streptavidin HRP together with DAB substrate and BrdU⁺ cells were counted for quantification.

Animal tissues were collected, fixed in 10% buffered formalin, and paraffin embedded. Immunohistochemical analyses were performed using a Ki67-specific antibody (Thermo RM-9106-S) following manufacturer’s protocols. BrdU (Life Technologies, 00–0103) labelling was detected using BrdU in situ detection kit (BD, 550803). Single molecule RNA in situ hybridization was done using QuantiGene ViewRNA Assays (Affymetrix) following manufacturer's instructions.

BrdU incorporation assay was done using BrdU In-Situ Detection Kit (550803, BD Pharmingen) according to the manufacturer’s instructions. In brief, 2 hr prior to euthanasia mice were injected intraperitoneally with BrdU (B5002, Sigma Aldrich), at a dose of 50 mg/kg body weight of mice. Tissue samples were fixed in 4% paraformaldehyde and embedded in paraffin. The paraffin-embedded liver tissue sections were de-paraffinized by washing with xylene 2 times for 5 min each time at room temperature. The tissue sections were dehydrated by incubation in 100% ethanol 2 times for 5 min followed by once in 95% ethanol for 3 min at room temperature. The tissue sections were treated with 0.3% H₂O₂ to block endogenous peroxidase, followed by antigen retrieval with ‘BD Retrievagen A’ (#550803, BD Pharmingen) in a microwave oven to 89°C for 10 min. The tissue sections were incubated in biotinylated anti-BrdU antibody (#550803, BD Pharmingen) at 1:10 in diluent buffer for 1 hr at room temperature, and then in HRP-conjugated streptavidin for 1 hr at room temperature. The tissue sections were stained with DAB substrate solution followed by hematoxylin counterstaining. Slides were examined by bright field microscopy.