Xenograft tumor tissue samples from Nod-SCID mice were collected, fixed in 4% paraformaldehyde, and embedded in paraffin. The embedded samples were sliced into 4 μm-thin sections, which were deparaffinized, dehydrated, and subjected to antigen retrieval by immersing in a citrate buffer at 98°C for 15 min to block endogenous peroxidase activity. Thereafter, samples were subjected to H2O2 treatment and blocked with a 3% bovine serum albumin (BSA) solution for 30 min. The samples were then hybridized with antibodies against glutathione peroxidase 4 (GPX4; 1:100, bm5231, Boster), GOT1 (1:100, bm5408, Boster), LC3B (1:2000, ab51520, Abcam), FTH1 (1:100, bm5356, Boster), NCOA4 (1:200, a5695, Abclonal), or 4HNE (1:25, ab51520, Abcam). After 12 h, the slices were incubated with the corresponding secondary antibodies and then subjected to staining using a 3, 3’-diaminobenzidine (DAB) staining kit (Vector Laboratories, USA).
Ab51520
Manufactured by Abcam
Sourced in United Kingdom, United States, China
Anti-HMGB1 antibody [EPR3507] (ab51520) is a recombinant rabbit monoclonal antibody that recognizes HMGB1 protein. The antibody is suitable for Western blotting, immunohistochemistry, and ELISA applications.
Automatically generated - may contain errors
Lab products found in correlation
Ab109012, by Abcam (16 mentions)
Ab56416, by Abcam (12 mentions)
Ab207612, by Abcam (11 mentions)
Ab8227, by Abcam (11 mentions)
Ab62557, by Abcam (10 mentions)
Pvdf membrane, by Merck Group (10 mentions)
Ab181602, by Abcam (8 mentions)
Ab32503, by Abcam (8 mentions)
Ab6721, by Abcam (8 mentions)
Ab8245, by Abcam (7 mentions)
Ab205718, by Abcam (6 mentions)
Ab9485, by Abcam (6 mentions)
Bca protein assay kit, by Beyotime (5 mentions)
Ab108327, by Abcam (4 mentions)
Ab23707, by Abcam (4 mentions)
Ab13847, by Abcam (4 mentions)
Ab91526, by Abcam (4 mentions)
Ripa lysis buffer, by Beyotime (4 mentions)
Ab210498, by Abcam (4 mentions)
Ripa lysis buffer, by Thermo Fisher Scientific (4 mentions)
122 protocols using ab51520
1
Immunohistochemical Analysis of Xenograft Tumors
2
Immunohistochemical and Immunofluorescence Analysis
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Immunohistochemical and immunofluorescence analyses of samples were performed as described. Rabbit anti-SIRT1 (sc15404, Santa Cruz Biotechnology, Santa Cruz, CA, United States), rabbit anti-FOXO3a antibody (12829, Cell Signaling Technology, CA, United States), rabbit anti-α-SMA antibody (ab32575, Abcam, Cambridge, MA, United States), rabbit anti-LC3II antibody (ab51520, Abcam, Cambridge, MA, United States), rabbit anti-SQSTM1/p62 (ab109012), and fluorescein isothiocyanate-labeled goat anti-rabbit IgG (sc-2012, Santa Cruz Biotechnology, Santa Cruz, CA, United States) were used in the immunostaining assay for kidney tissues. Rabbit anti-FOXO3a antibody (12829, Cell Signaling Technology, CA, United States), rabbit anti-SIRT1 (2977886, Millipore, Billerica, MA, United States), rabbit anti-LC3II antibody (ab51520, Abcam, Cambridge, MA, United States), rabbit anti-SQSTM1/p62 (ab109012), rabbit anti-α-SMA antibody (ab32575, Abcam, Cambridge, MA, United States), and Alexa Fluor 488 donkey anti-rabbit IgG (Invitrogen, Carlsbad, CA, United States) were used for immunostaining the cells. The nuclei of the cells were stained with DAPI. Immunopositive signals were detected using a confocal microscope (Leica Microsystems, Wetzlar, Germany) or an Olympus BX60 microscope (Olympus, Tokyo, Japan). The acquired images were analyzed using the IPP 6.0 software.
3
Immunofluorescence Staining of Autophagy Markers
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Primary HESCs, human peritoneal macrophages, THP-1 cells, and HESCs after multiple treatments were fixed in ice-cold methanol for 15 min, washed three times with 4°C PBS, incubated for 15 min in 0.25% Triton X-100 diluted in PBS, immersed in PBS twice for 3 min, and blocked with 1% BSA in PBS for 30 min at about 26°C. The primary HESCs and HESCs were incubated with anti-Beclin1 (1:100; ab62557, Abcam), anti-LC3B (1:100; ab51520, Abcam), SQSTM1/p62 (1:500; ab109012, Abcam), and ULK1 (1:200; ab203207, Abcam). Human peritoneal macrophages and THP-1 cells were incubated with anti-MST1 (1:400; ab51134, Abcam) and anti-p38-MAPK (1:200; ab170099, Abcam). The incubation time of each antibody varied from 1 h to 24 h at 4°C. All antibodies were diluted in 1% BSA in PBS. After cells were immersed in PBS twice for 3 min, they were incubated with the anti-mouse/rabbit secondary antibody for 30 min at about 26°C. Anti-quench nuclear staining and mounting with DAPI (ab104139, Abcam, USA) were performed. Images were captured with a confocal microscope (LSM880 Airy, Zeiss) and processed with the Zen software (Zeiss).
4
Evaluating Autophagy in PBMC
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Microtubule-associated protein light chain 3 (LC3B) is an autophagosome membrane marker so we employed immunocytochemistry with anti-LC3B to evaluate the occurrence of autophagy, on PBMC (Klionsky et al., 2021 (link)). Calls were fixed with paraformaldehyde 4% for 20min at 8°C and permeabilized. Cells were subsequently stained with anti-LC3B (ab51520 Abcam, EUA) and the secondary rat anti-IgG labeled with Alexa Fluor® 488 (Invitrogen, EUA), and were observed under the fluorescence microscope. Images were acquired using a digital camera (Coolpix 990, Nikon). Results were represented after counting at least 100 cells in each experimental time point using the software ImageJ®.
5
Western Blot Analysis of Cellular Proteins
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Twenty-five micrograms of total protein extracts prepared according to standard methods were fractioned by SDS-PAGE (4–12% Bis–Tris/SDS polyacrylamide gel, NuPAGE, Thermo Fisher Scientific) and transferred onto Hybond nitrocellulose filters (GE Healthcare Life Sciences, Buckinghamshire, UK). Filters were blocked for 1 h at room temperature in 1× PBS-Tween20, 5% skim milk, and then incubated overnight at 4 °C with primary antibodies: Rabbit polyclonal anti-γ-H2AX (ab11174, Abcam, Cambridge, UK); anti-LAMP-1 (ab24170, Abcam); anti-LC3B (ab51520, Abcam); and anti-PARP-1 (#9542, Cell Signaling Technology, Danvers, MA, USA). Mouse monoclonal anti-GAPDH (G8796, Sigma-Aldrich S.r.l.) and anti-Vinculin (VCL, V9131, Sigma-Aldrich S.r.l.) antibodies were used to ensure equal protein loading. The filters were then probed with secondary, peroxidase-linked whole antibodies (GE Healthcare) for 1 h at room temperature, and blotted proteins were detected using the Novex® ECL HRP Chemiluminescent detection system (Thermo Fisher). Filters were then subjected to autoradiography, the films were scanned, and images were analyzed using ImageJ 1.46r.
6
Quantification of Autophagy-Related Proteins
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The brain sections were submerged in citrate buffer for antigen retrieval. The protein levels of LC3B (1:100, ab51520, Abcam), Beclin1 (1:200, 11306-1-AP, Proteintech), p62 (1:200, ab109012, Abcam) and PINK1 (1:100, 23274-1-AP, Proteintech) were examined in each group after primary antibody incubation overnight at 4 °C and secondary antibody incubation at 37 °C for 45 min. DAPI was used to dye the nuclei. The sections were then viewed under a fluorescence microscope (Olympus). Images were captured at a magnification of 400X.
7
Autophagy Protein Quantification
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Anti-LC3B antibody 1:3000 (Abcam, ab51520), Anti-HDAC3 antibody 1:4000 (Abcam, ab32369), Tip60 Antibody 1:1000 (Cell Signaling, #12058), Beclin-1 Antibody 1:1000 (Cell Signaling, #3738), and Anti-ULK1 antibody 1:5000 (Abcam, ab128859) were utilized for experiments.
8
Immunofluorescence Staining of LC3B
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4.5–5 × 104 cells were seeded onto a Nunc Lab-Tek II Chamber Slide (Thermo Scientific, Waltham, MA, USA) and allowed to adhere overnight at 37 °C. The following day, cells were fixed with ice-cold methanol: acetone (1:1) for 10–15 min. The fixed cells were rinsed three times with washing buffer (0.1% Tween-20 in 1X PBS) and non-specific binding site was blocked with blocking buffer (10% goat serum diluted in washing buffer) for 1 h at room temperature. The cells were incubated for 2 h with anti-LC3B antibody (ab51520, Abcam, Cambridge, UK; 1:1000) diluted in blocking buffer. Following three 10 min washes in washing buffer, the slides were incubated with AlexaFluor 488-conjugated goat anti-rabbit secondary antibody (A11034, Thermo Fisher Scientific, Waltham, MA, USA; 1:1000) diluted in blocking buffer for 1 h in room temperature. The slides were further washed in washing buffer three times, 10 min each in the dark and mounted with Vectashield antifade mounting medium with DAPI (Vector Laboratories, Burlingame, CA, USA). The slides were observed under a confocal microscope (LSM 510 META, Zeiss, Oberkochen, Germany) and the staining analysed using ImageJ software55 (link).
9
NLRP1 Activation and Autophagy Signaling
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The NLRP1 activator muramyl dipeptide (MDP) (Selleck, USA) was then added at 100 μmol for 24 hours to the oxidative stress model according to the preliminary experiment and the published studies [21 (link)–24 (link)]. The cell viability determination was the same as that in Section 2.1 .
Total protein from HTR-8/SVneo cells was prepared with RIPA lysing buffer. The sample proteins (20 μg) of the different groups were separated using 10% SDS-PAGE and transferred onto PVDF membranes. These membranes were incubated with a primary antibody, followed by incubation of the anti-rabbit IgG secondary antibody. Protein expression was detected with an enhanced chemiluminescence detection kit. GAPDH served as an internal control. The antibodies included those for IL-1β (Abcam, ab216995), pro-IL-1β (Abcam, ab216995), pro-CASP1 (Abcam, ab207802), CASP1 (Abcam, ab207802), Beclin-1 (Abcam, ab207612), LC3 (Abcam, ab51520), p62 (Abcam, ab211324), ATG5 (Abcam, ab108327), ATG7 (Abcam, ab52472), NLRP3 (Abcam, ab263899), NLRP1 (BioLegend, 679802), and GAPDH (Abcam, ab128915). An ECL chemiluminescent reagent and imaging system (Bio-Rad, USA) were used to display protein bands, with the collected images analyzed using Bio-Rad software.
Total protein from HTR-8/SVneo cells was prepared with RIPA lysing buffer. The sample proteins (20 μg) of the different groups were separated using 10% SDS-PAGE and transferred onto PVDF membranes. These membranes were incubated with a primary antibody, followed by incubation of the anti-rabbit IgG secondary antibody. Protein expression was detected with an enhanced chemiluminescence detection kit. GAPDH served as an internal control. The antibodies included those for IL-1β (Abcam, ab216995), pro-IL-1β (Abcam, ab216995), pro-CASP1 (Abcam, ab207802), CASP1 (Abcam, ab207802), Beclin-1 (Abcam, ab207612), LC3 (Abcam, ab51520), p62 (Abcam, ab211324), ATG5 (Abcam, ab108327), ATG7 (Abcam, ab52472), NLRP3 (Abcam, ab263899), NLRP1 (BioLegend, 679802), and GAPDH (Abcam, ab128915). An ECL chemiluminescent reagent and imaging system (Bio-Rad, USA) were used to display protein bands, with the collected images analyzed using Bio-Rad software.
10
Immunohistochemical Analysis of Autophagy Markers
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Paraffin-embedded tissue sections were cut to 100 μm thick, dried, deparaffinized and rehydrated following standard protocols. The sections were incubated with primary antibodies against Beclin1 (1:500; ab622557; Abcam, Cambridge, MA, USA), LC3 (1:2,000; ab51520; Abcam) and LAMP2 (1:1,000; ab25631; Abcam) at 4°C overnight. Then, the sections were incubated with secondary antibodies for 30 min at room temperature. The sections were stained by diaminobenzidine (DAB; Thermo Fisher Scientific, Waltham, MA, USA). Nuclei were lightly stained with hematoxylin. For negative control, the sections were treated as above but PBS (Hyclone, South Logan, UT, USA) instead of primary antibodies.
For each section, three fields were randomly selected (×200). The expression scores of Beclin1, LC3, and LAMP2 were on the grounds of staining intensity (no coloring: 0 point; light yellow: 1 point; brown yellow: 2 points; sepia: 3 points) and percentage of positive tumor cells (0–5%: 0 point; 6–25%: 1 point; 26–50%: 2 points; >50%: 3 points) (17 (link)). The final score was determined by staining intensity score × percentage of positive tumor cells (>4 scores: positive and 0–3 scores: negative).
For each section, three fields were randomly selected (×200). The expression scores of Beclin1, LC3, and LAMP2 were on the grounds of staining intensity (no coloring: 0 point; light yellow: 1 point; brown yellow: 2 points; sepia: 3 points) and percentage of positive tumor cells (0–5%: 0 point; 6–25%: 1 point; 26–50%: 2 points; >50%: 3 points) (17 (link)). The final score was determined by staining intensity score × percentage of positive tumor cells (>4 scores: positive and 0–3 scores: negative).
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