Spinal cord tissue sections were treated with blocking solution (10% BSA and 2% Triton X-100, 1:1) for 2 h and incubated with primary antibodies (rabbit anti-rat LC3, ab128025, Abcam; mouse anti-rat NeuN, MAB377, Merck Millipore) at 4°C overnight. PC12 cells were fixed in 4% paraformaldehyde-0.1 mol/l PBS solution, treated with 10% normal goat serum for 1 h, and incubated with rabbit anti-rat LC3 (ab128025, Abcam) at 4°C overnight. After PBS washing, the sections were incubated with fluorescein-labeled secondary antibodies (goat anti-rabbit IgG Alexa Fluor 488, ab150077, Abcam; goat anti-mouse IgG Alexa Fluor 647, ab150115, Abcam) at room temperature in the dark for 2 h; the cells were incubated with goat anti-rabbit IgG Alexa Fluor 488 (ab150077, Abcam) at room temperature in the dark for 2 h. Nuclei were stained with DAPI. Images were captured using a fluorescence microscope (TCS SP2, Leica) and imported into ImageJ for processing. The channels were split, and the channels of the two target proteins were reserved. False colors were added to the two channels which were processed using the Coloc 2 plug-in.
Ab128025
Manufactured by Abcam
Sourced in United States, United Kingdom
Ab128025 is a recombinant monoclonal antibody that recognizes an epitope on the human CD274 (PD-L1) protein. This antibody can be used for immunohistochemistry (IHC) applications.
Automatically generated - may contain errors
Lab products found in correlation
Ab56416, by Abcam (12 mentions)
Ab207612, by Abcam (7 mentions)
Ab109012, by Abcam (7 mentions)
Ab181602, by Abcam (6 mentions)
Ab6721, by Abcam (6 mentions)
Ab91526, by Abcam (6 mentions)
Pvdf membrane, by Merck Group (6 mentions)
Ab8227, by Abcam (5 mentions)
Ab8245, by Abcam (5 mentions)
Ab210498, by Abcam (5 mentions)
Ab49822, by Abcam (4 mentions)
Ab9485, by Abcam (4 mentions)
Ab184787, by Abcam (3 mentions)
L0668, by Merck Group (3 mentions)
Ab6276, by Abcam (3 mentions)
Ab133448, by Abcam (3 mentions)
Protein assay kit, by Beyotime (3 mentions)
Ab62557, by Abcam (3 mentions)
Ab205718, by Abcam (3 mentions)
Ab32572, by Abcam (3 mentions)
62 protocols using ab128025
1
Immunofluorescence Analysis of Autophagy and Neuronal Markers
2
Immunohistochemical Analysis of Xenografts
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Xenografts of Huh-7 with THP-1 derived macrophages were fixed with 4% neutral paraformaldehyde. Next, the paraffin-embedded sections (4 μm in thickness) were prepared for hematoxylin and eosin (H&E) staining and immunohistochemical staining of LC3 (ab128025, Abcam, Cambridge, UK). The steps of immunohistochemical staining were described above. The pretreatment of H&E staining was basically the same as the immunohistochemical steps. Sections were treated with hematoxylin reagent for 5 min after deparaffinization and rehydration and then treated with 1% acid–ethanol for 1 s. Subsequently, the sections were stained by eosin reagent for 3 min. The slides were dehydrated and mounted then photographed by microscopy.
3
Western Blot Analysis of Autophagy and Apoptosis
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Western blot assay was performed following the standard protocol. In brief, after total protein was isolated and quantified, the proteins were separated by sodium dodecyl sulfonate-polyacrylamide gel (SDS-PAGE; Solarbio, Beijing, China) and then transferred onto polyvinylidene difluoride membranes (PVDF; Pall Corporation, New York, NYC, USA). Next, the membranes were blocked in slim milk for 2 h and then incubated with primary antibodies against ATG3 (ab108251; 1:2000; Abcam, Cambridge, MA, USA), LC3Ⅱ/LC3Ⅰ (ab128025; 1:200; Abcam), P62 (ab56416; 1:500; Abcam), B-cell lymphoma-2 (BCL-2; ab196495; 1:1000; Abcam), cleaved caspase-3 (ab49822; 1:500; Abcam), cleaved caspase-9 (ab2324; 1:2000; Abcam), BCL2-Associated X (Bax; ab182733; 1:2000; Abcam) or GAPDH (ab181602; 1:5000; Abcam) at 4°C overnight followed by incubation with HRP-conjugated secondary antibody (D110150; 1:5000; Sangon, Shanghai, China) for 2 h. The protein bands were analyzed using an enhanced chemiluminescence reagent (Beyotime).
4
Immunofluorescent Analysis of Spinal Cord Tissue
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The spinal cord tissues of each group were fixed with 4% paraformaldehyde at 4 °C. The spinal cord tissue was placed at the bottom of 50 mL centrifuge tube and dehydrated in 10%, 20%, and 30% sucrose solution. Twelve micrometers frozen sections were made and detected. All sections were blocked with blocking solution [10% goat serum, 3% bovine serum albumin (BSA), and 0.1% Triton X-100] for 1 h at 37 °C and then incubated overnight at 4 °C with Rabbit-anti-Active-Caspase3 (1:500, ab49822, Abcam), Rabbit-anti-Caspase3 (1:500, ab184787, Abcam), Rabbit-anti-LC3 (1:100, ab128025, Abcam), Rabbit-anti-LAMP2 (1:400, L0668, Sigma), Rabbit-anti-Bcl-2 (1:500, NB100-56101, NOVUS) and Rabbit-anti-Bax (1:500, ab12503, Abcam); 0.01 M phosphate buffer saline (PBS) was used to wash them for 10 min at 3 times and followed by incubating with a mixture of FITC- or Cy3-conjugated secondary antibodies for 2 h at room temperature and then being washed again with PBS for 10 min at 3 times. The stained sections were examined with a Leica fluorescence microscope (Leica DM 5000B, Germany).
5
Antibody Profiling for Mitochondrial Dynamics
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The following antibodies were used: anti-Fundc1 polyclonal antibody (P050, AVIVA), anti-Nix polyclonal antibody (CST12396, Cell Signaling Technology), anti-Bcl2L13 polyclonal antibody (16612, Proteintech), anti-Bnip3 polyclonal antibody (#3769, Cell Signaling Technology), anti-Drp1 monoclonal antibody (611113, BD), anti-Mff polyclonal antibody (17090-1-AP, Proteintech), anti-Fis1 polyclonal antibody (10956-1-AP, Proteintech), anti-Actin monoclonal antibody (A5441, Sigma Aldrich), anti-LC3B antibody (PM036, Medical and Biological Laboratories Co.), anti-p-Drp1(S616) antibody (#3455, Cell Signaling Technology), anti-Tim23 antibody (FNab08693, FineTest), Anti-LC3A/B antibody for immunofluorescence (ab128025, Abcam), donkey anti-rabbit IgG (H + L) highly cross-adsorbed secondary antibody, Alexa Fluor 594 (A21207, Invitrogen Thermo Fisher Scientific), Alexa Fluor 488 donkey anti-rabbit IgG (A21206, Invitrogen Thermo Fisher Scientific), SSEA-1 (SC-21702AF647, Santa Cruz Biotechnology).
6
Immunohistochemical analysis of tumor microenvironment
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Representative H&E‐stained glass slides containing intratumoral lesions were examined. A core (3 mm in size) was collected from the invasive tumor front of each representative paraffin block and transplanted into the recipient tumor microarray (TMA) blocks. Immunohistochemical staining was carried out using an automated immunostainer (Benchmark Ultra, Ventana Medical Systems Inc., Tucson, AZ, USA) with monoclonal antibodies: anti‐CD9 antibody at a dilution of 1:50 (ab2215, Abcam, Cambridge, MA, USA, anti‐CD63 antibody at a dilution of 1:500 (ab134045, Abcam, Cambridge, MA, USA), anti‐LC3A/B antibody at a dilution of 1:100 (ab128025, Abcam, Cambridge, MA, USA), anti‐P62 antibody at a dilution of 1:2000 (ab56416, Abcam, Cambridge, MA, USA), and anti‐ANXA1 antibody at a dilution of 1:100 (ab33061, Abcam, Cambridge, MA, USA).
7
Immunoblotting for Autophagy Markers
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Immunoblotting was carried out as described previously [16 (link)]. The following antibodies were employed in this study: Anti-ATG7 (1:2000 dilution), anti-ATG12 (1:1000), LC3 A/B (1:500), and anti-β-actin (1:2000) (cat. ## ab133528, ab155589, ab128025, and ab6276, respectively, Abcam). After blocking in 5% BSA/PBS containing 0.1% of Tween 20 for 1 h, these membranes were probed with the primary antibodies overnight at 4 °C. After incubation with a secondary antibody (1:3000), chemiluminescent signals were registered and scanned, and the band intensity was quantified using ImageJ software. β-actin was used always as the internal loading control.
8
Immunoblotting Analysis of Autophagy and Apoptosis Markers
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MLO-Y4 cells were homogenized in lysis buffer, and protein concentration was quantified using the Bradford assay. After separation through 15% SDS-PAGE and transferred onto PVDF membranes, cell lysate was subjected to immunoprecipitation with specific antibodies for 1 h at RT, followed by immunoblotting according to the kit’s protocols (Roche, Basel, Switzerland). The primary antibodies used for immunostaining were: rabbit anti-JNK1 (1:1000, Abcam, ab199380), rabbit anti-p-JNK1 ab47337, rabbit anti-AMPK (1:1000, Abcam, ab32382), rabbit anti-p-AMPK (1:1000, Abcam, ab133448), rabbit anti-Beclin 1(1:1000, Abcam, ab207612), rabbit anti-Bcl-2 (1:1000, Abcam, ab182858), rabbit anti-LC3 (1:1000, Abcam, ab128025), rabbit anti-Cleaved Caspase-3 (1:1,000, Abcam, ab32042), rabbit anti-Cleaved PARP (1:1000, Abcam, ab32064), rabbit anti-SOD1 (1:1000, Abcam, ab179843), rabbit anti-SOD2 (1:1000, Abcam, ab74231), rabbit anti-Actin (1:3000, Abcam, ab8227). The secondary antibodies used for immunostaining were: mouse HRP (1:2000, Abcam, ab6728), rabbit HRP (1:2000, Abcam, ab6721).
9
Western Blotting and Immunohistochemistry Protocols
10
Quantitative Protein Analysis in Cells
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Total protein content was extracted with the help of a radioimmunoprecipitation assay lysis buffer (P0013B, Beyotime Biotechnology, Shanghai, China) containing 1% protease inhibitor and phosphorylase inhibitor, and the obtained protein concentration was analyzed using bicinchoninic acid kits (Boster, A53226, Thermo Fisher Scientific). After undergoing electrophoresis separation, the proteins were transferred onto a polyvinylidene fluoride membrane (IPVH85R, Merck Millipore, Billerica, MA). Following blockade with 5% bovine serum albumin (BSA), the membrane was probed with primary antibodies (Abcam Inc., Cambridge, UK) against HDAC8 (ab187139, 1 : 10000), IRF1 (ab191032, 1 : 1000), SUCNR1 (ab272856, 1 : 1000), LC3 (ab128025, 1 : 1000), and GAPDH (ab181602, 1 : 2500, normalizer) overnight at 4°C. The following day, the membrane was reprobed with the horseradish peroxidase- (HRP-) labeled secondary antibody IgG (ab6721, 1 : 5000, Abcam) for 2 h. Afterwards, the immunocomplexes were visualized with an enhanced chemiluminescence reagent, and band intensities were quantified using the ImageJ 1.48u software (V1.48, National Institutes of Health, Bethesda, Maryland).
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