Figure 1. Western blot analysis of Lipocalin 2 using anti-Lipocalin 2 antibody (MBS178180).
Electrophoresis was performed on a 5-20% SDS-PAGE gel at 70V (Stacking gel) / 90V (Resolving gel) for 2-3 hours. The sample well of each lane was loaded with 50ug of sample under reducing conditions.
Lane 1: Mouse Lung Tissue Lysate,
Lane 2: Mouse Intestine Tissue Lysate.
After Electrophoresis, proteins were transferred to a Nitrocellulose membrane at 150mA for 50-90 minutes. Blocked the membrane with 5% Non-fat Milk/ TBS for 1.5 hour at RT. The membrane was incubated with rabbit anti-Lipocalin 2 antigen affinity purified polyclonal antibody at 0.5ug/mL overnight at 4 degree C, then washed with TBS-0.1%Tween 3 times with 5 minutes each and probed with a goat anti-rabbit IgG-HRP secondary antibody at a dilution of 1:10000 for 1.5 hour at RT. The signal is developed using an Enhanced Chemiluminescent detection (ECL) kit with Tanon 5200 system. A specific band was detected for Lipocalin 2 at approximately 22KD. The expected band size for Lipocalin 2 is at 22KD.
Figure 2. IHC analysis of Lipocalin 2 using anti-Lipocalin 2 antibody (MBS178180).
Lipocalin 2 was detected in paraffin-embedded section of Mouse Spleen Tissue. Heat mediated antigen retrieval was performed in citrate buffer (pH6, epitope retrieval solution) for 20 mins. The tissue section was blocked with 10% goat serum. The tissue section was then incubated with 1ug/ml rabbit anti-Lipocalin 2 Antibody (MBS178180) overnight at 4 degree C. Biotinylated goat anti-rabbit IgG was used as secondary antibody and incubated for 30 minutes at 37 degree C. The tissue section was developed using Strepavidin-Biotin-Complex (SABC) with DAB as the chromogen.
Figure 3. IHC analysis of Lipocalin 2 using anti-Lipocalin 2 antibody (MBS178180).
Lipocalin 2 was detected in paraffin-embedded section of Rat Spleen Tissue. Heat mediated antigen retrieval was performed in citrate buffer (pH6, epitope retrieval solution) for 20 mins. The tissue section was blocked with 10% goat serum. The tissue section was then incubated with 1ug/ml rabbit anti-Lipocalin 2 Antibody (MBS178180) overnight at 4 degree C. Biotinylated goat anti-rabbit IgG was used as secondary antibody and incubated for 30 minutes at 37 degree C. The tissue section was developed using Strepavidin-Biotin-Complex (SABC) with DAB as the chromogen.
Figure 4. IHC analysis of Lipocalin 2 using anti-Lipocalin 2 antibody (MBS178180).
Lipocalin 2 was detected in frozen section of mouse lung tissue. Heat mediated antigen retrieval was performed in citrate buffer (pH6, epitope retrieval solution) for 20 mins. The tissue section was blocked with 10% goat serum. The tissue section was then incubated with 1ug/ml rabbit anti-Lipocalin 2 Antibody (MBS178180) overnight at 4 degree C. Biotinylated goat anti-rabbit IgG was used as secondary antibody and incubated for 30 minutes at 37 degree C. The tissue section was developed using Strepavidin-Biotin-Complex (SABC) with DAB as the chromogen.
Figure 5. IHC analysis of Lipocalin 2 using anti-Lipocalin 2 antibody (MBS178180).
Lipocalin 2 was detected in frozen section of mouse spleen tissue. Heat mediated antigen retrieval was performed in citrate buffer (pH6, epitope retrieval solution) for 20 mins. The tissue section was blocked with 10% goat serum. The tissue section was then incubated with 1ug/ml rabbit anti-Lipocalin 2 Antibody (MBS178180) overnight at 4 degree C. Biotinylated goat anti-rabbit IgG was used as secondary antibody and incubated for 30 minutes at 37 degree C. The tissue section was developed using Strepavidin-Biotin-Complex (SABC) with DAB as the chromogen.
Figure 6. IHC analysis of Lipocalin 2 using anti-Lipocalin 2 antibody (MBS178180).
Lipocalin 2 was detected in frozen section of rat lung tissue. Heat mediated antigen retrieval was performed in citrate buffer (pH6, epitope retrieval solution) for 20 mins. The tissue section was blocked with 10% goat serum. The tissue section was then incubated with 1ug/ml rabbit anti-Lipocalin 2 Antibody (MBS178180) overnight at 4 degree C. Biotinylated goat anti-rabbit IgG was used as secondary antibody and incubated for 30 minutes at 37 degree C. The tissue section was developed using Strepavidin-Biotin-Complex (SABC) with DAB as the chromogen.
Figure 7. IHC analysis of Lipocalin 2 using anti-Lipocalin 2 antibody (MBS178180).
Lipocalin 2 was detected in frozen section of rat spleen tissue. Heat mediated antigen retrieval was performed in citrate buffer (pH6, epitope retrieval solution) for 20 mins. The tissue section was blocked with 10% goat serum. The tissue section was then incubated with 1ug/ml rabbit anti-Lipocalin 2 Antibody (MBS178180) overnight at 4 degree C. Biotinylated goat anti-rabbit IgG was used as secondary antibody and incubated for 30 minutes at 37 degree C. The tissue section was developed using Strepavidin-Biotin-Complex (SABC) with DAB as the chromogen.
Western blot analysis of Lipocalin 2 using anti-Lipocalin 2 antibody. Electrophoresis was performed on a 12% SDS-PAGE gel at 80V (Stacking gel) / 120V (Resolving gel) for 2 hours. The sample well of each lane was loaded with 30 ug of sample under reducing conditions.
Lane 1: rat lung tissue lysates,
Lane 2: mouse lung tissue lysates.
After electrophoresis, proteins were transferred to a nitrocellulose membrane at 150 mA for 50-90 minutes. Blocked the membrane with 5% non-fat milk/TBS for 1.5 hour at RT. The membrane was incubated with rabbit anti-Lipocalin 2 antigen affinity purified polyclonal antibody at 0.5 ug/mL overnight at 4°C, then washed with TBS-0.1%Tween 3 times with 5 minutes each and probed with a goat anti-rabbit IgG-HRP secondary antibody at a dilution of 1:5000 for 1.5 hour at RT. The signal is developed using an ECL Plus Western Blotting Substrate with Tanon 5200 system. A specific band was detected for Lipocalin 2 at approximately 23 kDa. The expected band size for Lipocalin 2 is at 23 kDa.
IHC analysis of Lipocalin 2 using anti-Lipocalin 2 antibody. Lipocalin 2 was detected in a paraffin-embedded section of mouse spleen tissue. Heat mediated antigen retrieval was performed in EDTA buffer (pH 8.0, epitope retrieval solution). The tissue section was blocked with 10% goat serum. The tissue section was then incubated with 2 ug/ml rabbit anti-Lipocalin 2 Antibody overnight at 4°C. Peroxidase Conjugated Goat Anti-rabbit IgG was used as secondary antibody and incubated for 30 minutes at 37°C. The tissue section was developed using HRP Conjugated Rabbit IgG Super Vision Assay Kit with DAB as the chromogen
IHC analysis of Lipocalin 2 using anti-Lipocalin 2 antibody. Lipocalin 2 was detected in a paraffin-embedded section of mouse spleen tissue. Heat mediated antigen retrieval was performed in EDTA buffer (pH 8.0, epitope retrieval solution). The tissue section was blocked with 10% goat serum. The tissue section was then incubated with 2 ug/ml rabbit anti-Lipocalin 2 Antibody overnight at 4°C. Peroxidase Conjugated Goat Anti-rabbit IgG was used as secondary antibody and incubated for 30 minutes at 37°C. The tissue section was developed using HRP Conjugated Rabbit IgG Super Vision Assay Kit with DAB as the chromogen.
IHC analysis of Lipocalin 2 using anti-Lipocalin 2 antibody. Lipocalin 2 was detected in a paraffin-embedded section of rat spleen tissue. Heat mediated antigen retrieval was performed in EDTA buffer (pH 8.0, epitope retrieval solution). The tissue section was blocked with 10% goat serum. The tissue section was then incubated with 2 ug/ml rabbit anti-Lipocalin 2 Antibody overnight at 4°C. Peroxidase Conjugated Goat Anti-rabbit IgG was used as secondary antibody and incubated for 30 minutes at 37°C. The tissue section was developed using HRP Conjugated Rabbit IgG Super Vision Assay Kit with DAB as the chromogen.
IHC analysis of Lipocalin 2 using anti-Lipocalin 2 antibody. Lipocalin 2 was detected in a paraffin-embedded section of rat spleen tissue. Heat mediated antigen retrieval was performed in EDTA buffer (pH 8.0, epitope retrieval solution). The tissue section was blocked with 10% goat serum. The tissue section was then incubated with 2 ug/ml rabbit anti-Lipocalin 2 Antibody overnight at 4°C. Peroxidase Conjugated Goat Anti-rabbit IgG was used as secondary antibody and incubated for 30 minutes at 37°C. The tissue section was developed using HRP Conjugated Rabbit IgG Super Vision Assay Kit with DAB as the chromogen.
Effects of Nrf2 on MCTR1-regulated ferroptosis in CLP-induced AKI. Mice were given ML385 (30 mg/kg, ip, qd) for 7 d. On day 7, ML385 injection with or without a twice-daily administration mode of MCTR1 as described before in the following experiment. All kidney samples were collected at 24 h after CLP. A–D Shown are representative western blotting and quantification of Nrf2, GPX4, and PTGS2. E – G Quantitative analyses of MDA, GSH, and non-heme iron. H Quantitative analyses of Perl’s stain. I Representative Perl’s stain images. J Representative TEM images. The black arrow indicates ferroptosis-like mitochondria. K Quantitative analyses of mitochondrial length. L Histological analyses of renal tubular injury. M , N Representative HE stains and PAS stain. O Representative IHC images for NGAL. P Quantitative analyses of IHC stain of NGAL. Q , R Quantitative analyses of serum Scr and BUN. n?=?6 mice/group, mean ± SD were presented. **P < 0.01
Effects of MCTR1 on ferroptosis in CLP-induced AKI. Mice were given MCTR1 once, twice, or not during the 24 h experiment. The once-daily administration mode (qd): Mice were given MCTR1 (200 ng/mice, iv) 0.5 h before being subjected to CLP. The twice-daily administration mode (bid): Mice were given MCTR1 (200 ng/mice, iv) 0.5 h before being subjected to CLP, and then an additional MCTR1 (200 ng/mice, iv) was given 12 h after CLP. All kidney samples were collected at 24 h after CLP. A , B Shown are representative western blotting and quantification of GPX4 and PTGS2. C–E Quantitative analyses of MDA, GSH, and non-heme iron. F Quantitative analyses of Perl’s stain. G Representative Perl’s stain images. H Representative TEM images. The black arrow indicates ferroptosis-like mitochondria. I Quantitative analyses of mitochondrial length. J Histological analyses of renal tubular injury. K , L Shown are representative HE stains and PAS stain. M Representative IHC images for NGAL. N Quantitative analyses of IHC stain of NGAL. O , P Quantitative analyses of serum Scr and BUN. n?=?6 mice/group, mean ± SD were presented. **P < 0.01 . iv: intravenous
Ferroptosis is present in CLP-induced AKI. Mice were subjected to CLP, then the kidneys were collected at the indicated time. A , B Shown are representative western blotting and quantification of GPX4 and PTGS2. C–E Quantitative analyses of MDA, GSH, and non-heme iron. F Quantitative analyses of Perl’s stain. G Representative Perl’s stain images. H Representative transmission electron microscopy (TEM) images. The black arrow indicates ferroptosis-like mitochondria. I Quantitative analyses of mitochondrial length. Mice were pretreated with liproxstatin-1 (10 mg/kg, ip) or DFO (20 mg/kg, ip) 0.5 h before being subjected to CLP. All kidney samples were collected at 24 h after CLP. J–K Shown are representative Hematoxylin-eosin (HE) stains and Periodic Acid-Schiff (PAS) stain. L Histological analyses of renal tubular injury. M Quantitative analyses of IHC stain of NGAL. N Representative immunohistochemistry (IHC) images for NGAL. O , P Quantitative analyses of Scr and BUN. n?=?6 mice/group, mean ± SD were presented. *P < 0.05, **P < 0.01 and ns : P > 0.05 . ip: intraperitoneal
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