Formalin fixed, paraffin-embedded tissue blocks from the investigated cases were evaluated including the following anatomical regions: frontal, anterior cingulate, parietal, temporal, occipital cortex, hippocampus, entorhinal cortex, amygdala, basal ganglia, thalamus, mesencephalon, pons, medulla oblongata, and cerebellum. In addition to hematoxylin and eosin (HE) and Gallyas silver staining, the following mouse monoclonal antibodies were used for immunohistochemistry: anti-p62 (1:1000+MW; Abnova), anti-tau PHF-1 (Ser396/Ser404, 1:2000; Gift of Peter Davies), anti 4R (RD4, 1:10000+MW; Millipore) and 3R (RD3, 1:1000+FA; Millipore) tau isoforms; anti-phospho-TDP-43 (rat monoclonal TAR5P-1D3; pS409/410 TDP-43; Ascenion, Munich, Germany; gift of Manuela Neumann)(33 (link)), anti-α-synuclein (Syn303, 1:30000+FA; CNDR), anti-Aµ (Nab228, 1:20000; CNDR) and anti-FUS (fused in sarcoma; 1:8000+MW; ProteinTech). The Vectashield ABC detection kit, peroxidase/DAB, rabbit/mouse/rat (BA1000/BA2000/BA4001, 1:1000; Vector Laboratories) was used for the visualization of antibody reactions. Neuropathological alterations (astrogliosis/neuronal loss, degree of various protein depositions) were semi-quantitatively (none, mild, moderate, severe) evaluated in different anatomical regions.
Anti p62
Manufactured by Abnova
Sourced in Germany
Anti-p62 is a laboratory product used for the detection and quantification of the p62 protein in biological samples. p62 is a ubiquitin-binding protein that plays a role in the autophagic degradation of proteins. This product can be used as a tool for research purposes, but its intended use should not be extrapolated.
Automatically generated - may contain errors
Lab products found in correlation
Anti cleaved caspase 3, by Cell Signaling Technology (2 mentions)
Anti gapdh, by Abcam (2 mentions)
Anti lc3, by Merck Group (2 mentions)
Anti lc3, by Cell Signaling Technology (2 mentions)
Anti lc3, by Novus Biologicals (2 mentions)
Polyvinylidene fluoride membrane, by Merck Group (1 mentions)
Anti tubulin, by Merck Group (1 mentions)
Anti flag, by Merck Group (1 mentions)
Anti ha, by Cell Signaling Technology (1 mentions)
Anti tomm20, by Santa Cruz Biotechnology (1 mentions)
Anti caveolin 1, by Abcam (1 mentions)
Anti timm23, by BD (1 mentions)
Anti beclin 1, by Novus Biologicals (1 mentions)
Anti vps34, by Cell Signaling Technology (1 mentions)
Anti lc3b, by Novus Biologicals (1 mentions)
Odyssey system, by LI COR (1 mentions)
Diaminobenzidine dab, by Vector Laboratories (1 mentions)
Methyl green, by Vector Laboratories (1 mentions)
Vectastain elite abc kit, by Vector Laboratories (1 mentions)
Anti lc3, by Nanotools (1 mentions)
14 protocols using anti p62
1
Comprehensive Neuropathological Analysis of Neurodegeneration
2
Immunoprecipitation and Immunoblot Analysis
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The primary antibodies used include anti-LC3B, anti-beclin1 (Novus Biologicals, Littleton, CO, USA), anti-caveolin1 (Abcam, Cambridge, UK), anti-TIMM23, anti-phospho-caveolin1 (BD Bioscience, San Jose, CA, USA), anti-HA, anti-Vps34 (Cell Signaling Technology, Danvers, MA, USA), anti-tubulin, anti-Flag (Sigma-Aldrich), anti-p62 (Abnova, Taiwan) and anti-TOMM20 (Santa Cruz Biotechnology, Dallas, TX, USA). Immunoprecipitation assays were performed as previously described;6 (link) briefly, cells were lyzed with modified RIPA buffer (10 mM Tris-HCl, 150 mM NaCl, 5 mM EDTA, 1 mM Na3VO4, 1% CHAPS). After pull-down with the appropriate antibodies, same amounts of protein were separated by SDS–polyacrylamide gel electrophoresis (PAGE) and transferred onto the polyvinylidene fluoride membrane (Millipore, Billerica, MA, USA). Immunoblot analysis was then performed and visualized by the enhanced chemiluminescence method.
3
Multi-Modal Analysis of Neurodegeneration in Mice
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Anesthetized mice were subjected to intracardiac perfusion with 4% paraformaldehyde in PBS. Whole brains were removed and post-fixed in 4% paraformaldehyde and subsequently embedded in paraffin. Four-µm-thick sections were mounted on glass slides. Antigen retrieval was performed manually using either a high pH Tris or citric-acid based solution (Vector Labs) at 95°C for 10 minutes. IHC staining was performed with anti-HSV antigen (Dako) diluted 1∶5000, anti-LC3 (Nanotools) diluted 1∶400, anti-cleaved caspase-3 (Cell Signaling) diluted 1∶500, or anti-p62 (Abnova) diluted 1∶2000 with the Vectastain Elite ABC kit (Vector Labs). HRP labeled secondary antibodies were visualized after treatment with the chromagen diaminobenzidine (DAB, Vector Labs). Finally, the slides were washed in tap water, counterstained in Gill's Hematoxylin, and imaged with the EVOS XL core cell imaging system. Western blots were performed on whole brain homogenates using a 1∶1000 dilution of anti-beclin 1 antibody (BD Biosciences) and anti- GAPDH (Abcam) as a loading control. Blots were visualized and densitometry analysis was performed using the LI-COR Odyssey system.
Paraffin-embedded sections were assayed for DNA fragmentation using the TUNEL technique (EMD Millipore) and counterstained with Methyl Green (Vector Labs). H&E staining was performed using Gill's Hematoxylin and Eosin Y solution.
Paraffin-embedded sections were assayed for DNA fragmentation using the TUNEL technique (EMD Millipore) and counterstained with Methyl Green (Vector Labs). H&E staining was performed using Gill's Hematoxylin and Eosin Y solution.
4
Immunostaining Analysis of Autophagy Markers
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Cells were fixed with 4% paraformaldehyde and permeabilized in PBS with 0.1% BSA (Sigma, A4503) and 0.1% saponin (Sigma, 84510). Immunostaining was performed using an anti-Ku70 antibody (Santa Cruz, sc-17789), anti-ATG5 ab (Sigma, A0856), and anti-p62 (Abnova, H00008878-M01) and anti-LC3 (Sigma, L8918). As secondary antibodies anti-mouse IgG Alexa Fluor 488 (Invitrogen, catalog no. A11001), anti-rabbit IgG Alexa Fluor 488 (Invitrogen, catalog no. A11008), anti-rabbit IgG Alexa Fluor 568 (Invitrogen, catalog no. A11011) were used. Cells also were co-stained with Hoechst for 10 min. (BD 33,442). Cover slides were mounted and inspected under 63× magnification using a Carl Zeiss LSM 710 confocal microscope (Zeiss, Germany).
5
Mitochondrial Profiling and Protein Analysis
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Mitochondrial and cytosolic fractions were isolated using a commercial kit (Qiagen, Germany). For whole‐cell lysates, cells were collected in RIPA buffer (20 mM Tris‐HCl, 150 mM NaCl, 1 mM EDTA, 1% NP40, 0.1% SDS, antiphosphates and antiproteases) and centrifuged at 13,000 g for 5 min to obtain the supernatant. Equal amounts of proteins were electrophoresed and transferred to a nitrocellulose membrane. Primary antibody incubations were performed with anti‐MAO‐A or anti‐parkin from Abcam; anti‐p53, anti‐phospho‐p53(ser15), anti‐H2A.X, anti‐pink1, anti‐LC3, anti‐ubiquitin, anti‐phospho‐Rb(Ser807/811), anti‐phospho‐p70S6K(Thr389) from Cell Signaling Technologies; anti‐phospho‐H2A.X(Ser139) from Millipore; anti‐p62 from Abnova; and anti‐ATM, anti‐phospho‐ATM(Ser1981), anti‐p21 from Santa Cruz Biotechnology. Images were taken with the ChemiDoc‐MP Imaging System and quantified using Image‐Lab 4.0 software (Bio‐Rad).
6
Western Blot Analysis of OXPHOS Proteins
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Antibodies anti‐COX4, anti‐UQCRC2, anti‐NDUFA9, anti‐ATP5A, anti‐SDHA were from Abcam (OXPHOS cocktail ab110412, dilution 1:2,000); anti‐SDHB (ab14714, dilution 1:200) and anti‐COX1 (ab14705, dilution 1:2,000) were from Abcam; anti‐HSC70 was from Santa Cruz (sc‐7298, dilution 1:1,000); anti‐GAPDH was from Abcam (ab8245, dilution 1:40,000); anti‐P62 was from Abnova (H00008878‐M01, dilution 1:1,000); anti‐LC3‐I/II was from Novus Biologicals (NB100‐2220, dilution 1:1,000); anti‐LAMP1 was from Cell Signaling (3243, dilution 1:1,000); anti‐PINK1 was from Novus (BC100‐494, dilution 1:1,000); and anti‐Parkin was from Abcam (ab77924, dilution 1:500).
7
Immunofluorescence Analysis of Autophagy Markers
8
Tau and Autophagy Protein Detection
9
Immunoblotting for APP, BACE1, and Oxidative Stress
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Anti-C20 (1:1000) antibody used to detect APP and its β-CTFs was kindly provided by laboratory of Professor Weihong Song. Anti-BACE1 (1:1000, ab183612) and anti-PS1 (1:1000, ab76083) antibodies were purchased from Abcam (Cambridge, MA, USA). Anti-Ubquitin (1:1000, #10201-2-AP), anti-Beclin-1 (1:1000, #11306-1-AP), anti-Nrf2 (1:1000, #10396-1-AP), anti-HO-1(1:1000, #10701-1-AP), anti-SOD-1 (1:1000, #10269-1-AP), and anti-caspase-3 (1:1000, #66470-2-Ig) antibodies were obtained from Proteintech (Wuhan, Hubei, China). Anti-P62 (1:1000, H00008878-M01), anti-LC3 (1:1000, #12741) and anti-β-actin (1:3000, A5411) antibodies were purchased from Abnova (Taipei, Taiwan, China), Cell Signaling Technology (Danvers, MA, USA) and Sigma (St. Louis, MO, USA), respectively.
10
Antibody Analysis of Autophagy Regulators
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The following antibodies were used in this study: anti-LC3 (#3868, Cell Signaling Technology), anti-YKT6 (sc-365732, Santa Cruz Biotechnology), anti-vinculin (700062, Thermo Fisher Scientific), anti-p62 (H00008878-M01, Abnova), anti-SNAP29 (ab181151, Abcam), anti-NDP52 (ab68588, Abcam), anti-OPTN (HPA003360, Sigma Aldrich), anti-TOM20 (sc-17764, Santa Cruz Biotechnology), anti-HaloTag (G9211, Promega), anti-ULK1 (#8054, Cell Signaling Technology), anti-FIP200 (#12436, Cell Signaling Technology). All antibodies were used at a 1:1000 dilution for western blotting and 1:200 for fluorescence microscopy. Uncropped images of western blots are shown in Fig. S5 .
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