Frozen kidney tissues were embedded in OCT and sliced into 6-μm-thick sections for immunofluorescence. Sections were fixed in 4% PFA for 15 min and then permeabilized using 0.5% Triton X-100 in PBS for 10 min. After blocking with 5% BSA in PBS for 1 h at room temperature, they were incubated with antibodies, anti-F4/80 (70076, CST), anti-CD206 (ab64693, Abcam), and anti-p-STAT3 Y705 (ab191419, Abcam) overnight at 4°C. The secondary antibodies used included Donkey anti-Rabbit IgG-AlexaFluor 488 (abs20020, Absin), Donkey anti-Rabbit IgG-Cy3 (abs20022, Absin), and Donkey anti-Mouse IgG-AlexaFluor 594 (abs20017, Absin). The nuclei were stained with Hoechst33258. Images were acquired using an Olympus FV3000 confocal microscope.
Ab191419
Manufactured by Abcam
Ab191419 is a lab equipment product from Abcam. It is a primary antibody used for immunohistochemistry (IHC) and immunocytochemistry (ICC) applications.
Automatically generated - may contain errors
Lab products found in correlation
Ab64693, by Abcam (3 mentions)
Fv3000 confocal microscope, by Olympus (2 mentions)
Ab32143, by Abcam (2 mentions)
Ab192029, by Abcam (2 mentions)
Ab233548, by Abcam (2 mentions)
Abs20017, by Absin (1 mentions)
Image lab software version 3, by Bio-Rad (1 mentions)
Ab7817, by Abcam (1 mentions)
Ab181602, by Abcam (1 mentions)
Peroxidase affinipure goat anti rabbit igg h l, by Jackson ImmunoResearch (1 mentions)
Peroxidase affinipure goat anti mouse igg h l, by Jackson ImmunoResearch (1 mentions)
Phrodo red s aureus bioparticles, by Thermo Fisher Scientific (1 mentions)
Cellmask green, by Thermo Fisher Scientific (1 mentions)
Hoescht 33342 nuclear stain, by Thermo Fisher Scientific (1 mentions)
Ab23680, by Abcam (1 mentions)
Ab3283, by Abcam (1 mentions)
Ab8227, by Abcam (1 mentions)
4 protocols using ab191419
1
Immunofluorescence Analysis of Kidney Tissue
2
Comprehensive Western Blot Analysis Protocol
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Western blot was performed as previously described (11 (link)). The primary antibodies used included anti-CD206 (ab64693, Abcam), anti-Arg-1 (ab233548, Abcam), anti-Ym1 (ab192029, Abcam), anti-SIRPα (ab191419, Abcam), anti-p-STAT3 S727 (ab32143, Abcam), anti-p-STAT3 Y705 (#9664, CST), anti-STAT3 (9662, CST), anti-α-SMA (ab7817, Abcam), anti-cleaved Caspase 3 (CST; 9662), anti-Caspase 3 (CST; #3498), anti-Bcl2 (CST; #3498), anti-E-cadherin (GTX100443, GTX), and anti-GAPDH (ab181602, Abcam). The secondary antibodies included Peroxidase AffiniPure Goat Anti-Rabbit IgG (H+L) and Peroxidase AffiniPure Goat Anti-Mouse IgG (H+L) (111035003, 115035003, Jackson, West Grove, PA, USA). Protein levels were quantified using the Image Lab software, version 3.0 (Bio-Rad, Hercules, CA, USA).
3
Antibody-based Assay Optimization
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The monoclonal antibodies for β-actin (ab8227), SIRP-α (ab191419), α-chimaerin (ab156869), CD47 (ab3283), and CD36 (ab23680) were purchased from Abcam (Cambridge, MA). The αvβ3 integrin antibody (LM609; mAb1976) and FLAG tag antibody (M2; F1804) were purchased from Millipore Sigma (Burlington, MA). A monoclonal antibody for GULP1/CED-6 (NB100–1042) was purchased from Novus Biologicals (Littleton, CO). IRDye800-conjugated secondary antibodies (rabbit and mouse) were purchased from Li-Cor Biosciences (Lincoln, NE). pHrodo™ Red S. aureus bioparticles, Hoescht 33342 nuclear stain, and CellMask Green were purchased from Invitrogen Life Technologies (Carlsbad, CA). siRNAs against human ITGB5, GULP1/CED6, ABR, and SIRPA and a non-targeting siRNA were purchased from Dharmacon (Lafayette, CO). The pcDNA3.1 plasmid expressing FLAG-tagged (DYKDDDK) CHN1 was purchased from GenScript (OHu26717D; Piscataway, NJ) and validated by DNA sequencing. The forward and reverse primers used for the sequencing were 5′-TAA TAC GAC TCA CTA TAG GG-3′ and 5′-CCT CGA CTG TGC CTT CTA-3′, respectively.
4
Immunophenotyping of Activated Macrophages
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Raw264.7 cells were mounted on slides, and treatments were performed. After treatment, slides were fixed in 4% PFA for 15 min and then permeabilized using 0.5% Triton X-100 in PBS for 10 min. After blocking with 5% BSA in PBS for 1 h at room temperature, they were incubated with antibodies, including anti-CD206 (ab64693, Abcam), anti-Arg-1 (ab233548, Abcam), anti-Ym1 (ab192029, Abcam), anti-SIRPα (ab191419, Abcam), and anti-p-STAT3 S727(ab32143, Abcam) overnight at 4°C. The secondary antibodies were as described above. The nuclei were stained with Hoechst33258. Images were acquired using an Olympus FV3000 confocal microscope.
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