After H290 and 8A cells were treated with PAL, they were then incubated for 8 h/37 °C. The cells were then fixed with 4% (w/v) paraformaldehyde for 10 min/RT, washed 3 times using PBS, incubated with 0.1% (v/v) Triton X100 and blocked for 1 h at RT with 3% (w/v) bovine serum albumin. The cells were then incubated overnight at 4 °C with primary antibodies against iNOS (Cat. #CXNFT; 1/200 dilution; Invitrogen), p65 (Cat. #D14E12; 1/400 dilution; Cell Signaling Technology), LAMP1 (Cat. #ab25630; 1/500 dilution; Abcam), LC3B (Cat. #D11; 1/1000 dilution; Cell Signaling Technology), SQSTM1/p62 (Cat. #D5L7G, 1/1000 dilution; Cell Signaling Technology), TFEB (Cat. #1337-I-AP, 1/100 dilution; Proteintech) and NF-κB (Cat. #PA5-88084, 1/500 dilution; Thermofisher), washed 3 times with PBS and incubated with the secondary antibodies, Alexa Fluor® Plus 488 or Alexa Fluor® Plus 568 (Invitrogen). A Zeiss confocal microscope (LSM880, Carl Zeiss) was used to observe cellular morphology. The fluorescence intensity and Mander's overlap for image co-localization were measured using ImageJ 4.7v software. Fifty cells were quantified with ImageJ software for integration of each fluorescence (wavelength) area via excluding the cellular background. Relative intensities per cell in arbitrary units are shown.
Pa5 88084
Manufactured by Thermo Fisher Scientific
The PA5-88084 is a high-performance laboratory equipment designed for various scientific applications. It is a compact and durable device that delivers consistent and reliable results. The core function of the PA5-88084 is to perform precise measurements and analysis within a controlled environment. Detailed specifications and intended use can be provided upon request.
Automatically generated - may contain errors
Lab products found in correlation
Ab125066, by Abcam (3 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Ab175186, by Abcam (2 mentions)
Ma5 42523, by Thermo Fisher Scientific (2 mentions)
Alexa fluor plus 488, by Thermo Fisher Scientific (1 mentions)
Lamp1, by Cell Signaling Technology (1 mentions)
Sqstm1 p62, by Cell Signaling Technology (1 mentions)
Alexa fluor plus 568, by Thermo Fisher Scientific (1 mentions)
Confocal microscope lsm880, by Zeiss (1 mentions)
Cxnft, by Thermo Fisher Scientific (1 mentions)
Ab76003, by Abcam (1 mentions)
Nuclear protein extraction kit, by Solarbio (1 mentions)
Gtx22770, by GeneTex (1 mentions)
Pierce fast western blot kit, by Thermo Fisher Scientific (1 mentions)
Ripa lysis buffer, by Beyotime (1 mentions)
Ab79199, by Abcam (1 mentions)
Ab205718, by Abcam (1 mentions)
Ab155282, by Abcam (1 mentions)
Image pro plus 7, by Media Cybernetics (1 mentions)
Dm2500, by Leica (1 mentions)
7 protocols using pa5 88084
1
Immunofluorescence Analysis of Autophagy Markers
2
Protein Extraction and Western Blot Analysis
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Total proteins were harvested from cells or liver tissues with RIPA Lysis Buffer (Beyotime, Shanghai, China) supplemented with phenylmethyl sulfonyl fluoride (PMSF) protease inhibitor and phosphatase inhibitor. A nuclear protein extraction kit (Solarbio, Beijing, China) was used to separate cytoplasmic and nuclear proteins. The corresponding protein concentration was determined by Pierce™ BCA Protein Assay Kit (Thermo Fisher, Massachusetts, USA). The denatured protein samples with appropriate protein amount were transferred to PVDF membrane after sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The membranes were then blocked with 5% skimmed milk for 1 h and incubated overnight with specific antibodies against MMP-9 (ab76003, Abcam), AhR (GTX22770, GeneTex), CYP1A1(GTX55582, GeneTex), Nrf2 (PA5-88084, Thermofisher), Lamin B1(YT5180, Immunoway), and GAPDH (ANT325, Antgene) in a shaker at 4 ℃. After washing and secondary antibody incubation (ANT019/020, Antgene), the protein bands were visualized by the FluorChem Imaging System (ProteinSimple, California, USA) using the commercial Pierce™ Fast Western Blot Kit (Thermo Fisher, Massachusetts, USA).
3
Spinal Cord Histology and Immunohistochemistry
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Coronal sections of spinal cord segments were fixed using 4% paraformaldehyde and subsequently embedded in paraffin. Paraffin-embedded sections were sliced (5 μm thick) and subjected to haematoxylin and eosin (H&E) and Nissl staining.
For immunohistochemical preparation, the sections were incubated with primary antibodies targeting pMST1 (phospho T183; 1:50; Ab79199; Abcam), NRF2(1:100; PA588084; Thermo), and SOD2 (1:200; 66474-1-IG; Proteintech) at 4 °C over-night. Subsequently, the sections were washed thrice with PBS and incubated with the respective secondary antibodies. The resulting reaction was visualized using DAB solution. After counterstaining with haematoxylin, the sections were examined under a light microscope (400 × magnification; Nikon, Tokyo, Japan).
For immunohistochemical preparation, the sections were incubated with primary antibodies targeting pMST1 (phospho T183; 1:50; Ab79199; Abcam), NRF2(1:100; PA588084; Thermo), and SOD2 (1:200; 66474-1-IG; Proteintech) at 4 °C over-night. Subsequently, the sections were washed thrice with PBS and incubated with the respective secondary antibodies. The resulting reaction was visualized using DAB solution. After counterstaining with haematoxylin, the sections were examined under a light microscope (400 × magnification; Nikon, Tokyo, Japan).
4
Immunohistochemical Analysis of NRF2, GPX4, and ACSL4
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Antigen was retrieved from paraffin NP tissue sections (4 μm) by boiling in EDTA buffer solution (0.05 mol/L Tris and 0.001 mol/L EDTA; pH 8.5) in a microwave oven for 20 min. The tissue sections were blocked with 5% BSA for 20 min and incubated with primary antibodies against NRF2 (1 : 200, PA5-88084, Thermo Fisher Scientific), GPX4 (1 : 200, ab125066, Abcam), and ACSL4 (1 : 200, ab155282, Abcam) overnight at 4°C, followed by incubation with biotinylated goat anti-rabbit IgG secondary antibody (1 : 2500, ab205718, Abcam) for 20 min. Thereafter, the sections were exposed to DAB substrate and counterstained with haematoxylin. Finally, the sections were photographed under a microscope (Leica-DM2500, Leica, Wetzlar, Germany). The ImagePro Plus 7.1 software (Media Cybernetics, Silver Spring, MD) was applied for quantitative analysis.
5
Protein Expression Analysis of Borax-Treated Cells
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Western blot analysis was performed to assess the protein levels of HSPA5, NRF2 and GPx4 in borax‐treated cells. Initially, protein samples (20 μg) were prepared and separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS‐PAGE). The separated proteins were then transferred from the gel to a polyvinylidene fluoride (PVDF) membrane. To prevent non‐specific binding, the PVDF membranes were blocked using 2% bovine serum albumin (BSA). Subsequently, the membranes were incubated overnight at 4°C with primary antibodies specific to HSPA5 (dilution 1 μg/mL; PA1‐014A; Invitrogen), NRF2 (dilution 1:2000; PA5‐88084; Invitrogen), GPx4 (dilution 1:1000; PA5‐102521; Invitrogen) and cleaved caspase‐3 (dilution 1:1000; Cat No. 9661; Cell Signaling). Following the primary antibody incubation, the membranes were washed to remove any unbound antibodies. Next, the membranes were incubated with secondary antibodies. To visualize the protein bands, a chemiluminescence ECL (enhanced chemiluminescence) kit (34579, Thermo Scientific) was used. The emitted light was captured using an imaging system, and the resulting images were analysed using software such as ImageJ.
6
Immunohistochemical Detection of Oxidative Stress Markers
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Prior to staining, kidney sections from rats underwent deparaffinization in xylene, which clears the paraffin from the sections, followed by a graded rehydration process. This typically involves a series of alcohol baths of decreasing concentration to gently reintroduce water into the tissues. Then the rehydrated sections were then incubated with the following primary antibodies for targeted detection of proteins: GPX-4 (1:100, rabbit monoclonal, ab125066, Abcam), xCT (1:200, rabbit monoclonal, ab175186, Abcam), ACSL4 (1:200, rabbit monoclonal, MA5-42523, Invitrogen), and Nrf-2 (1:100, rabbit polyclonal, PA5-88084, Invitrogen). The primary antibody incubation was performed in a controlled environment at 4 °C overnight to allow for maximum binding specificity and efficiency. Following primary antibody incubation, the sections were washed to remove unbound antibodies, and incubated with horseradish peroxidase (HRP)-conjugated secondary antibody. Finally, the HRP-DAB system (Proteintech, Wuhan, China) was applied to visualize the sections, followed by staining with hematoxylin to detect HRP activity. All sections were imaged under an Olympus BX53F microscope.
7
Protein Isolation and Western Blot Analysis
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Proteins were isolated from the collected cells and kidney samples using a radio-immunoprecipitation assay (RIPA) lysis buffer (R0010, Solarbio, Beijing, China). Following extraction, the protein levels were determined using a Bicinchoninic Acid (BCA) protein assay kit (PC0020, Solarbio, Beijing, China). The western blot analysis was conducted according to established methods. Primary antibodies utilized included GPX-4 (1:1000 dilution, ab125066, rabbit monoclonal, Abcam), xCT (1:1000 dilution, ab175186, rabbit monoclonal, Abcam), ACSL4 (1:1000 dilution, MA5-42523, rabbit monoclonal, Invitrogen), and Nrf-2 (1:1000 dilution, PA5-88084, rabbit polyclonal, Invitrogen). Secondary antibodies, used at a 1:5000 dilution, were acquired from Proteintech (Wuhan, China).
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