Total protein of PE embryonic tissues, EVs or transfected cells was extracted, and the bicinchoninic acid protein quantitative kit (ThermoFisher Scientific) was employed to determine protein concentration. Protein was electrophoretic separated by 10% sodium dodecyl sulphate polyacrylamide gels and transferred to nitrocellulose membranes (ZeYE Biological). The membrane was blocked with 5% skim milk powder and then placed in a 3% BSA blocking buffer. Then, membrane was probed with primary rabbit antibodies to Notch2 (1:1000, ab118824, Abcam), TIM3 (1:500, ab185703, Abcam), mTORC1 (1:1000, ab25880, Abcam), CD9 (1:2000, ab92726, Abcam), CD63 (1:1000, ab134045, Abcam), Calnexin (1:20 000, ab92537, Abcam) and β‐actin (1:1000, ab8227, Abcam), and then re‐probed with IgG antibody complexed to horseradish peroxidase (1:2000, ab97051, Abcam). Enhanced chemiluminescence solution (ECL808‐25, Biomiga) was added on membrane for colour development. After scanning and with an optical luminometer (GE, Pittsburg, USA), the protein bands were analysed by Image Pro Plus 6.0 software (Media Cybernetics, Silver Spring, Maryland, USA) to analyse the relative expression levels of proteins with β‐actin as the internal reference.
Ab185703
Manufactured by Abcam
Sourced in United Kingdom
Ab185703 is a laboratory product offered by Abcam. It is a high-quality reagent designed for use in research applications. The core function of this product is to provide researchers with a reliable tool for their experiments, but no further details about its intended use can be provided in an unbiased and factual manner.
Automatically generated - may contain errors
Lab products found in correlation
Image pro plus 6, by Media Cybernetics (1 mentions)
Optical luminometer, by GE Healthcare (1 mentions)
Bicinchoninic acid protein quantitative kit, by Thermo Fisher Scientific (1 mentions)
Ab118824, by Abcam (1 mentions)
Ab25880, by Abcam (1 mentions)
Ab134045, by Abcam (1 mentions)
Ab97051, by Abcam (1 mentions)
Ab92726, by Abcam (1 mentions)
Ab8227, by Abcam (1 mentions)
Ecl808 25, by Biomiga (1 mentions)
Cx41 microscope, by Olympus (1 mentions)
Q capture software, by Olympus (1 mentions)
Nbp2 30221, by Novus Biologicals (1 mentions)
A21207, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 488 labeled, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 594 dye, by Thermo Fisher Scientific (1 mentions)
A1r storm, by Nikon (1 mentions)
Fl 1031, by Vector Laboratories (1 mentions)
Anti 8 ohdg antibody, by Santa Cruz Biotechnology (1 mentions)
Fl 1321 2, by Vector Laboratories (1 mentions)
10 protocols using ab185703
1
Quantitative Protein Expression Analysis
2
Immunohistochemical Analysis of HAVCR2 and C10ORF54 in HCC
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Immunohistochemistry was performed as previously described (32 (link)). Briefly, paraffin embedded tissue slides with human HCC tissue microarray (TMA) (NBP2-30221, Novus Biologicals) were deparaffinized and rehydrated, endogenous peroxidise activity was blocked with 3% hydrogen peroxide, antigen retrieval was performed in 10 mmol/L citrate buffer, and nonspecific binding was blocked with blocking reagent. HAVCR2 (ab185703, Abcam) and C10ORF54 (CL3975, Invitrogen) antibodies were applied at 1:300 and 1:20 concentrations, respectively. Slides were incubated overnight at 4°C, followed by 30 min incubation with secondary anti-mouse or rabbit antibody HRP (Dako). The chromogen used was 3-amino-9-ethylcarbazole. Human normal and cancerous lung tissue was used as the positive control for both the antibodies and a negative control, for which the primary antibodies were substituted with the same concentration of mouse or rabbit IgG. Images were captured using a Olympus CX41 microscope and QCapture software. Immunohistochemical reactivity was evaluated by two independent investigators. The expression of HAVCR2 and C10ORF54 were categorized into positive staining or no staining.
3
Kidney Oxidative Stress and Co-Localization
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After dewaxing, rehydration, and blocking with BSA, the kidney cortex sections were treated with anti-8-OHdG antibody (Santa Cruz company, sc-66036) overnight at 4 °C. Then, the Alexa Fluor 488–labeled secondary antibody (A-11001, Thermo Fisher Scientific) was used. For detection of co-localization of Tim-3 and LTL/DBA, the primary antibodies were anti-Tim-3 antibody (1:100, ab185703, Abcam), anti-LTL antibody (1:200, FL-1321-2, Vector Laboratories), and anti-DBA antibody (1:100, FL-1031, Vector Laboratories), the secondary antibody was IgG antibody conjugated with Alexa Fluor 594 dye (1:1000, A21207, Thermo Fisher Scientific). After DAPI staining of nuclei, the sections were photographed under a fluorescence microscope (Nikon A1R Storm).
4
Cisplatin-Induced Kidney Injury Pathways
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BUMPT cells were stimulated with or without 40 μM cisplatin for about 24 h. SDS lysis buffer was used for protein extraction from BUMPT cells and kidney cortex tissues. Proteins were quantified by BCA quantification kit (CW0014, CWbiotech, China) and were separated by SDS–PAGE. After transferring to the PVDF membrane, proteins were incubated with primary antibodies overnight at 4 oC and secondary antibodies for about 2 h. The primary antibodies were anti-Bax antibody (2772, CST), anti-caspase-3 (9662, CST), anti-Bcl-2 antibody (ab59348, Abcam), anti-Tim-3 antibody (ab185703, Abcam), anti-HO-1 antibody (52947, Abcam), anti-pIKKα/β (phosphor S176/180) antibody (2697, CST), anti-NF-κB p65 (phosphor S536) antibody (ab86299, Abcam), anti-PARP antibody (BS7047, Bioword), anti-8-OHDG antibody (sc-66036, Santa Cruz), and anti-GAPDH antibody (AC033, Abclonal). The second antibodies were an anti-rabbit IgG antibody (BA1054, Boster) and an anti-mouse IgG antibody (BA1050, Boster). Chemiluminescent signals were analyzed using an ECL detection kit (WBKLS0500; Millipore). The densities of bands were measured with ImageJ software.
5
Immunostaining of CD8, CD56, TIM-3, Gal-9
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6
Immunohistochemical Analysis of Cell Markers
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Immunohistochemical staining was performed as described previously [55 (link)]. Briefly, coronal sections (4 μm thickness) were deparaffinized, rehydrated, and soaked in a 3% hydrogen peroxide (H2O2) solution for 10 min at room temperature to quench endogenous peroxidase activity. Next, sections were blocked in 5% bovine serum albumin for 30 min and were incubated overnight at 4 °C with the following primary antibodies: rabbit anti-Tim-3 (1:100, ab185703, Abcam), rabbit anti-Nrf2 (1:200, ab89443, Abcam), or rabbit anti-HMGB1(1:100, 3935S, Cell Signaling Technology). The next day, the sections were washed with PBS three times for 10 min each and were incubated with biotinylated goat anti-rabbit IgG (1:100, ZSGB-Bio, Beijing, China) for 30 min at room temperature. Sections were then incubated with horseradish peroxidase streptavidin for 10 min and developed with diphenylamine peroxidase substrate. Finally, sections were viewed and images were captured under a light microscope (Leica-DM2500, Leica Microsystems).
7
Multiplex Immunofluorescence Staining of FFPE Tissue
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For immunofluorescence staining, the slides are deparaffinized and rehydrated with graded ethanol and the process of antigen retrieval and non-specific binding blocking is the same as the previous IHC.
For the multiplexed immunofluorescence of CD4, CD8, PD-1, TIM3, TIGIT, and DAPI (for nuclear staining) in the FFPE tissue, we used a validated multiple fluorescence staining kit from Servicebio (Wuhan, China), according to the manufacturer’s recommendation. The following primary antibodies were used: mouse anti-human PD-1 (Abcam, ab52587, 1:200), rabbit anti-human TIGIT (Abcam, ab243903, 1:200), rabbit anti-human TIM3 (Abcam, ab185703, 1:400), and rabbit anti-human CD8 (Abcam, ab4055, 1:200). After the slides are incubated with each primary antibody and the corresponding HRP-labeled secondary antibody, the slides need to be labeled with different fluorescent dyes. The sample scanning, spectral unmixing, and quantification of signals were conducted with the panoramic MIDI Pathology Imaging System, using the CaseViewer V.2.3 software of 3DHISTECH.
For the multiplexed immunofluorescence of CD4, CD8, PD-1, TIM3, TIGIT, and DAPI (for nuclear staining) in the FFPE tissue, we used a validated multiple fluorescence staining kit from Servicebio (Wuhan, China), according to the manufacturer’s recommendation. The following primary antibodies were used: mouse anti-human PD-1 (Abcam, ab52587, 1:200), rabbit anti-human TIGIT (Abcam, ab243903, 1:200), rabbit anti-human TIM3 (Abcam, ab185703, 1:400), and rabbit anti-human CD8 (Abcam, ab4055, 1:200). After the slides are incubated with each primary antibody and the corresponding HRP-labeled secondary antibody, the slides need to be labeled with different fluorescent dyes. The sample scanning, spectral unmixing, and quantification of signals were conducted with the panoramic MIDI Pathology Imaging System, using the CaseViewer V.2.3 software of 3DHISTECH.
8
Immunohistochemical and Western Blot Analysis of BTK
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Immunohistochemical (IHC) analysis were conducted with standard procedures. BTK protein positive signals primarily located in the cell cytoplasm, which were counted according to the brown diaminobenzidine precipitate. The purified standard BTK (C481S, Human, full-length recombinant, MW: 77 kDa, Promega, Wisconsin, USA) as loading control. Western blotting was performed using the standard method. Briefly, after preparation of whole-cell lysates, proteins were separated through sodium dodecyl sulfate-polyacrylamide gel electrophoresis and were then transferred to polyvinylidene fluoride membranes. Specific primary antibodies used were anti-BTK (1:600, sc-81159; Santa Cruz), anti-T cell immunoglobulin-3 (TIM-3) (1:1000, ab185703, rabbit; Abcam), CD133 (1:1000, ab19898, rabbit; Abcam), vimentin (1:1000, ab137321, rabbit; Abcam), and anti-phospho-Btk (Tyr223) (1:1000, #5082, rabbit mAb; Cell Signaling). Anti-GAPDH (1:10000, sc-47724, Mouse mAb; Santa Cruz) served as the loading control.
9
Immunostaining of Kidney Macrophages and Podocytes
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For immunostaining of macrophages and podocytes, 3 μm thick formalin fixed, paraffin embedded kidney sections were stained with anti-Tim-3 antibody (ab185703, 1:200, Abcam, Southampton, UK), anti-Tim-3 (60355-1-Ig, 1:200, Proteintech, Wuhan, China), anti-CD68 antibody (ab955, 1:100, Abcam, Southampton, UK), anti-Nephrin antibody (ab136894, 1:200, Abcam, Southampton, UK), anti-WT-1 antibody (ab212951, 1:500, Abcam, Southampton, UK), anti-phosphorylated P65 NF-κB antibody (3033S, 1:100, CST, MA, USA) as primary antibody and anti-mouse and anti-rabbit (PV-9000, 1:200, ZSBIO, Beijing, China) as secondary antibody. Images were captured using an optical microscope. The final analysis of the images was performed using the software Image Pro Plus6.0.
10
Immunohistochemical Staining of Tim-3
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Tim-3 was immunohistochemically stained using a previously described standard technique (Li et al., 2018 (link)). Briefly, slides were deparaffinized in xylene and rehydrated in graded alcohol. Antigen retrieval was performed in tris-ethylenediaminetetraacetic acid (EDTA; pH 9.0) buffer at 95°C for 20 min. Slides were incubated in tris-buffered saline (TBS) for 5 min. Endogenous peroxidase blocking was performed in 3% H2O2 for 10 min. Subsequently, the slides were incubated in a rabbit polyclonal antibody against Tim-3 (1:500; Abcam, Inc., Cambridge, MA) overnight at 4°C. The slides were rinsed five times with 0.01 M phosphate-buffered saline (PBS; pH 7.4) for 10 min. Sections were incubated with primary antibodies against Tim-3 (1:1,000; catalog no. ab185703, Abcam) and with a horseradish peroxidase (HRP)–tagged secondary antibody (1:1,000; catalog no. sc-3836, Santa Cruz Biotechnology, Inc.) for another 1 hour at 37°C. Subsequently, the slides were washed in PBS and stained with 3,3-diaminobenzidine (DAB). Finally, the slides were counterstained, dehydrated, and mounted.
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