Immunofluorescence staining was performed as previously described.20 (link) Briefly, the cells cultured on cover slips were fixed, permeabilized with 0.5% Triton X-100, and incubated with anti-CD163 and anti-CD206 polyclonal antibodies (Proteintech, China. Lot: 00091171 and 00089604, respectively) (1:500) overnight at 4°C. Subsequently, the slides were incubated with secondary antibodies conjugated with Alexa Fluor 488 AffiniPure goat anti-mouse IgG (H+L) (FcMACS, China. Lot: 136908), goat anti-mouse IgG (H+L) CoraLite594 (Proteintech, China. Lot: 20000154), and goat anti-rabbit IgG (H+L) R-PE conjugate (Proteintech, China. Lot: 20000129) (1:1000). Nuclei were stained using 4ʹ,6-diamidino-2-phenylindole (DAPI) for 3 min, and the cells were incubated in the dark for 3 min. The slides were washed with PBS four times, for 5 min each. Then, the slides were sealed with sealing solution containing a fluorescence quencher and observed and imaged under a fluorescence microscope. Immunofluorescence was examined under an epi-fluorescence microscope (Olympus, BX60-32FB2-A03). By changing the filters, the different secondary antibodies could be identified in double-stained sections. Images were captured with a digital camera (Olympus, DP50).
Anti cd163
Manufactured by Proteintech
Sourced in China, United States
Anti-CD163 is a laboratory reagent used to detect the presence of the CD163 protein. CD163 is a cell surface glycoprotein expressed on monocytes and macrophages. The Anti-CD163 reagent can be used in various research and diagnostic applications to identify and study cells expressing CD163.
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Lab products found in correlation
Anti tgf β1, by Proteintech (2 mentions)
Anti gapdh, by Proteintech (2 mentions)
Goat anti mouse igg h l coralite594, by Proteintech (1 mentions)
Bx60 32fb2 a03, by Olympus (1 mentions)
Aperio at2, by Leica (1 mentions)
Anti il 1β, by Proteintech (1 mentions)
Goat anti rabbit igg, by Wuhan Servicebio Technology (1 mentions)
Anti mmp9, by Proteintech (1 mentions)
2 4 amidinophenyl 6 indolecarbamidine dihydrochloride dapi, by Wuhan Servicebio Technology (1 mentions)
Mouse anti cd68, by Thermo Fisher Scientific (1 mentions)
Anti tnfα, by ZenBio (1 mentions)
Anti il10, by Proteintech (1 mentions)
Anti ocn, by Affinity Biosciences (1 mentions)
Anti sdhb, by Proteintech (1 mentions)
Anti hif 1α, by Proteintech (1 mentions)
Anti inos, by Abcam (1 mentions)
Anti e cadherin, by Proteintech (1 mentions)
Anti vimentin, by Proteintech (1 mentions)
Anti cd68, by Thermo Fisher Scientific (1 mentions)
Anti akt, by Cell Signaling Technology (1 mentions)
7 protocols using anti cd163
1
Immunofluorescence Staining for CD163 and CD206
2
Immunohistochemical Analysis of Inflammatory Markers
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For immunohistochemical staining, after antigen retrieval, the slides were blocked with a 5% BSA solution for 1 hour and incubated with mouse anti-CD68 (1:100, Invitrogen, USA), anti-iNOS (1:100, Abcam, UK), anti-CD163 (1:1000, Proteintech, USA), anti-TNFα (1:100, ZenBio, China), anti-IL1β (1:100, Proteintech, USA), anti-IL10 (1:100, Proteintech, USA), anti-TGFβ1 (1:100, Proteintech, USA), anti-MMP9 (1:100, Proteintech, USA), anti-OCN (1:100, Affinity, China), anti-HIF1α (1:100, Proteintech, USA) and anti-SDHB (1:100, Proteintech, USA) at 4 °C overnight. Goat anti-rabbit IgG (1:100, Servicebio, China) was used as a secondary antibody and the nuclei were stained using 2-(4-amidinophenyl)-6-indolecarbamidine dihydrochloride (DAPI, Servicebio, China). Images were captured using Aperio AT2 (Leica, German).
3
Immunohistochemical Analysis of Placental Tissues
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For IHC, serial sections from the human placental villous tissues and the decidual tissues were obtained. IHC was conducted as previously described.24 The following primary antibodies were used: anti‐CD163 (Proteintech), anti‐Vimentin (Proteintech), anti‐G‐CSF (Abcam, USA) and anti‐E‐cadherin (Proteintech). Five visual fields were selected and observed in each section, and cells counted at 200 × magnification in each case using average values.
4
Immunophenotyping of Co-cultured Cells
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The HMC3 cells, after co-culture with T98G cells, were digested and seeded on the cell climbing slices. After washing with PBS, the slices were fixed with 4% paraformaldehyde and permeabilized with 0.3% Triton X-100. Then splices were blocked with BSA and incubated with anti-CD68 (ThermoFisher, US, 14-0688-82), anti-CD163 (Proteintech, US, 16646-1-AP) overnight and with fluorescent-labeled secondary antibodies for 90 min. The nucleus was stained with DAPI, and splices were sealed using glycerin. PBS washing was performed before each step above for 5 min, three times. The splices were observed under the fluorescent microscope. The experiments were repeated independently three times.
5
Investigating Akt, PI3Kγ, and SGK1 Signaling
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The mouse recombinant LIGHT protein (rLIGHT, cat#HY-P73837), PI3Kγ specific inhibitor (Eganelisib, IPI549, cat# HY-100716), and SGK1 inhibitor (GSK650394, cat#HY-15192) were purchased from MedChemExpress (MCE, New Jersey, USA). The primary antibodies anti-phospho-Akt (Ser473) (cat#4060) and anti-Akt (cat#4691)used for western blotting were purchased from Cell Signalling Technology (Danvers, Essex County, MA, USA); anti-PIK3CG (cat#A0266), anti-SGK1 (cat#A3936), and anti-phospho-Smad2/Smad3 (cat#AP1343) were purchased from ABclonal (Wuhan, China); anti-Smad2 (cat#ET1604-2), anti-Smad3 (cat#ET1607-4) and anti-PI3K p85α (cat# ET1608-70)were purchased from HUABIO (Hangzhou, China); and anti-F4/80 (cat#28463-1-AP), anti-CD3 (cat#17617-1-AP), anti-CD163 (cat#16646-1-AP), anti-ARG1 (cat#66129-1-Ig), anti-CD206(cat#60143-1-Ig), anti-iNOS (cat#18985-1-AP), anti-MCP1 (cat#66272-1-Ig), anti-TGFβ1 (cat#21898-1-AP), anti-collagen I (cat#14695-1-AP), anti-collagen III (cat#68320-1-Ig), anti-α-SMA (cat#14395-1-AP), anti-β actin (cat#20536-1-AP), and anti-GAPDH(cat#10494-1-AP) were purchased from Proteintech (Wuhan, China).
6
Hepatic Protein Analysis by Western Blot
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Proteins of hepatic samples or cells were analyzed using standard western blotting techniques. The antibodies of anti-α-SMA (ab32575, Abcam), anti-TGF-β1 (SAB4502954, Sigma), anti-P65 (10,745–1-AP, Proteintech), anti-p-P65 (AF2006, Affinity), anti-iNOS (13120S, CST), anti-CD86 (19,589, CST), anti-CD163 (16,646–1-AP, Proteintech), anti-CD206 (24,595, CST), anti-TLR4 (19,811–1-AP, Proteintech), anti-NLRP3 (A5652, ABclonal), anti-AKT (10,176–2-AP, Proteintech), anti-p-AKT (4060S, CST), anti-HO-1 (43,966, CST), anti-SOD1 (10,269–1-AP, Proteintecch), anti-MDA (MDA11-S, ADI), or anti-CASP1 (24232S, CST) were used as primary antibodies. Anti-GAPDH (10,494–1-AP, Proteintech) or anti-TUBULIN (#2144, CST) was used to normalize the signals. Bands were quantified by ImageJ software.
7
Quantifying Immune Cell Markers via Immunofluorescence
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The positive rate and intensity of protein expression were detected by immunofluorescence. The primary antibodies were anti-CD11B (Cat# 66519-1-Ig, ProteinTech, Wuhan, China) and anti-CD163 (Cat# 16646-1-AP, ProteinTech), and Alexa Fluor 488 Dnk secondary antibody was used for visualization (Cat# A21206 for anti-Rabbit, Cat# A21202 for anti-Mouse, Invitrogen, Carlsbad, CA, USA). The mean fluorescence intensity (MFI) of in situ immunofluorescence was assayed by the ImageJ program (Version 1.48v, Wayne Rasband, USA).
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