Protein lysates were prepared using RIPA buffer (Pierce, Rockford, IL, USA), and separated by gel electrophoresis. Proteins were then transferred onto PVDF membrane (Pierce). After blocking, the blots were incubated with appropriate primary and secondary antibodies. The protein bands were detected suing ECL reagents (Pierce). The intensities of bands were quantified using Image J software (NIH) and normalized with GAPDH. Primary antibodies used in this study: anti-ATF3 (1:1000, ab207434, Abcam, Cambridge, UK), anti-ILF3 (1:2000, ab131004, Abcam), anti-iNOS (1:1000, ab178945, Abcam), anti-Arg-1 (1:5000, PA529645, Invitrogen) and anti-GAPDH (1:2000, ab8245, Abcam) antibody.
Pa5 29645
Manufactured by Thermo Fisher Scientific
Sourced in United Kingdom
The PA5-29645 is a laboratory equipment product designed for general laboratory applications. It serves as a core function within the laboratory workflow. No further details can be provided while maintaining an unbiased and factual approach.
Automatically generated - may contain errors
Lab products found in correlation
Pa1 036, by Thermo Fisher Scientific (3 mentions)
Lsm 710 confocal microscope, by Zeiss (3 mentions)
Pa3030a, by Thermo Fisher Scientific (2 mentions)
Ab131004, by Abcam (1 mentions)
Ab207434, by Abcam (1 mentions)
Ab178945, by Abcam (1 mentions)
Ab8245, by Abcam (1 mentions)
Ab5076, by Abcam (1 mentions)
Ab131531, by Abcam (1 mentions)
Ma5 32255, by Thermo Fisher Scientific (1 mentions)
Rhodamine phalloidin, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 488 conjugated secondary antibody, by Thermo Fisher Scientific (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (1 mentions)
Ab45939, by Abcam (1 mentions)
Ab32575, by Abcam (1 mentions)
Ab28364, by Abcam (1 mentions)
Evos xl, by Thermo Fisher Scientific (1 mentions)
Ibright analysis software, by Thermo Fisher Scientific (1 mentions)
Ibright fl1000 imaging system, by Thermo Fisher Scientific (1 mentions)
Lps from e coli o55 b5, by Merck Group (1 mentions)
7 protocols using pa5 29645
1
Western Blot Analysis of Protein Targets
2
Multimarker Immunofluorescence Staining of Spinal Cord
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Deparaffinised spinal cord tissue sections were rehydrated and applied with 3% H2O2 solution and rinsed with PBS buffer. Antigen retrieval was done with applying 0.1 M sodium citrate solution. Then, sections were blocked with goat serum with a 30 min incubation at 37°C. Residual serum was removed by then. Primary antibodies against NeuN (#702022, Invitrogen, USA), iNOS (PA1-036, Invitrogen, USA), arg-1 (PA5-29645, Invitrogen, USA), IBA-1 (ab5076, Abcam, UK), NLRP3 (MA5-32255, Invitrogen, USA), and MK2 (ab131531, Abcam, UK) were applied to the sections followed by the overnight incubation at 4°C. After the removal of diluted primary antibody solutions, sections were rinsed with PBS buffer solution, ant incubated with fluorescent-labelled secondary antibody solution at 37°C for 30 min. DAPI staining solution was performed onto the sections for cell nucleus staining after thorough rinse with PBS buffer. Antifade reagent was applied to the sections after that to block the sections. Sections were then observed with a fluorescent microscope.
3
Quantifying Arginase 1 and Ki67 in VK2 Cells
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VK2 cells were fixed in 4% (v/v) paraformaldehyde in Dulbecco’s phosphate-buffered saline for 15 minutes at room temperature. Cells were permeabilised with 0.01% Triton X-100, blocked with 1% bovine serum albumin and incubated in rabbit anti-Arginase 1 (PA5-29645; Invitrogen, Thermo Fisher Scientific) or mouse anti-Ki67 (MM1, Leica, Milton Keynes, UK) primary antibodies overnight at 4C. Detection was achieved using Alexa Fluor 488 conjugated secondary antibodies (Invitrogen). Rhodamine phalloidin and DAPI (both Invitrogen) were used to counterstain the cytoskeleton and cell nuclei, respectively. Imaging was performed using an LSM 710 confocal microscope (Carl Zeiss, Cambridge, UK) with 405 nm diode, 488 nm argon and 561 nm diode lasers. Intensity of staining (corrected total cell fluorescence) (27 (link)) was measured from 30 cells per image, across 30 images per group (three cell passages). Cell counts were performed across entire frames in ImageJ software v.1.8.0 (National Institutes of Health, Bethesda, MD).
4
Immunohistochemical Characterization of Acellular Dermal Matrix
5
Quantitative Immunoblotting of Macrophage Markers
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BMMs at a concentration of 2 × 106 cells/well were adhered on 6-well plates overnight. The cells were treated for 24 h with the following stimuli: OMVs (10 μg per well), FSC200 (MOI = 50), LPS from E. coli O55:B5 (Sigma-Aldrich; 500 ng/mL), or IL-4 (Sigma-Aldrich; 20 ng/mL). Untreated BMMs were used as control. The cells were then washed twice with PBS and lysed with RIPA buffer for 30 min on ice and overnight at −80°C. The lysates were cleared by centrifugation (14,000 × g, 10 min, 4°C), and protein concentrations were determined by bicinchoninic acid assay. Samples were separated by 8% SDS-PAGE and electroblotted onto polyvinylidene difluoride (PVDF) membrane (Boehring). The proteins were immunodetected by primary rabbit polyclonal antibodies against alpha-tubulin 1A (#PA5-22060), iNOS (#PA3-030A), and arginase 1 (#PA5-29645, all Invitrogen), and polyclonal HRP-conjugated swine anti-rabbit immunoglobulins (Dako Cytochrom) were used as the secondary antibody. The reaction was visualized by SuperSignal™ West Femto Maximum Sensitivity Substrate on the iBright™ FL1000 Imaging System (both Thermo Fisher Scientific) and quantified with iBright Analysis Software. Local background corrected volumes were used and the values were normalized using alpha tubulin 1A bands as the house-keeping protein.
6
Nanomaterial Effects on Macrophage Polarization
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The RAW 264.7 cells were first treated with nanoparticles (20 μg/mL), followed by stimulation with LPS (100 ng/mL) under high glucose conditions for 24 h (for ICC and WB analysis). Subsequently, immunocytochemistry staining was performed using a general protocol. Briefly, the cells were fixed in 4% paraformaldehyde (PFA), permeabilized with Triton X-100, and blocked with a commercially available solution (DAKO). Dilutions of primary antibodies (supplementary data in Table S2 ) were prepared using 3% bovine serum albumin (BSA) (Sigma) in 0.1% Tween-phosphate-buffered saline (TPBS). For macrophage staining, primary antibodies against iNOS (1:100, rabbit, PA1-036, Thermo Fisher), IL-1b (1:100, rabbit, P420B, Thermo Fisher), and Arg-1 (1:200, rabbit, PA5-29645, Thermo Fisher) were treated overnight at 4 °C. After three washing steps, secondary antibodies, including F-actin (Alexa Fluor™ 488, Invitrogen), Rhodamine, and FITC conjugated (1:200, Jackson Immuno Research, Inc.), were treated for 1 h at room temperature (RT). DAPI was used for counterstaining. The IF staining sections were analyzed using a confocal laser scanning microscope (CLSM; Zeiss LSM 700 and IX71, Olympus), and the quantitative analysis was performed using ImageJ software.
7
Histological Evaluation of Intestinal Inflammation
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Colon sections were stained with hematoxylin and eosin (H&E) and graded for inflammation (0, none; 1, slight; 2, moderate; 3, severe), extent (0, none; 1, mucosa; 2, mucosa and submucosa; 3, transmural), crypt damage (0, none; 1, basal 1/3 damage; 2, basal 2/3 damage; 3, only surface epithelium lost; 4, entire crypt and epithelium lost), and percent of involvement (1, 1~25%; 2, 26~50%; 3, 51~75%; 4, 76~100%) as described37 (link). The score of each parameter was multiplied by a factor reflecting the percentage of tissue involvement to yield the final score. For evaluation using IHC, 5-μm-thick tissue sections were transferred onto adhesive slides and dried at 62 °C for 30 min. After incubation in 3% H2O2 to block the endogenous peroxidase activity, tissue sections were subjected to standard heat-mediated epitope retrieval in ethylene diamine tetraacetic acid (pH 8.0) for 32 min. The samples were incubated with primary antibodies against iNOS (1:50; PA1–036, Thermo Scientific, Rockford, IL), Arg-1 (1:100; PA5-29645, Thermo Scientific), and CD68 (1:2000; ab955, DAKO, Glostrup, Denmark), respectively. Immunostaining was detected using the DAKO IHC Detection Kit (K1492, DAKO). All IHC slides were counterstained with hematoxylin, dehydrated in ethanol, and cleared in xylene.
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