Renal tissue and cells were harvested and lysed by RIPA lysis buffer (Wuhan Goodbio Technology, China) containing cocktail protease inhibitors (Wuhan Goodbio Technology, China) for 30 min on ice. The total protein was obtained by high-speed centrifugation at low temperatures. Protein concentrations were quantified by a BCA protein assay kit (Beyotime Institute of Biotechnology, China), and 30 µg protein was loaded, separated on 10% or 12% SDS-PAGE, and transferred to nitrocellulose membranes. Then membranes were blotted at 4 °C overnight with mouse anti-VEGF-C (Santa Cruz, USA; 1:250), rabbit anti-Prox-1 (Angiobio, USA; 1:1000), rabbit anti-LYVE-1 (Novus, USA; 1:1000), Hamster anti-Podoplanin (Angiobio, USA; 1:200), rabbit anti-Collagen1 (Novus, USA; 1:2000), rabbit anti-α-SMA (Abcam, USA; 1:4000), rabbit anti-PDGFR-β (Abcam, USA; 1:2000), mouse anti-iNOS (Santa Cruz, USA; 1:200), rabbit anti-Arginase (Santa Cruz, USA; 1:400), rabbit anti-LC3B (Sigma-Aldrich, USA; 1:1000), rabbit anti-p62 (Abcam, USA; 1:5000), mouse anti-GAPDH (Wuhan Goodbio Technology, China; 1:2000), then were incubated with HRP-conjugated anti-IgG (Jackson ImmunoResearch, USA; 1:4000), finally were detected by ECL (Pierce, USA). Image capture and analysis were conducted by Bio-RAD (USA).
Rabbit anti p62
Manufactured by Abcam
Sourced in United States, United Kingdom
Rabbit anti-p62 is a primary antibody that recognizes the p62/SQSTM1 protein. p62/SQSTM1 is a multifunctional protein involved in various cellular processes such as autophagy and stress response. This antibody can be used for the detection and analysis of p62/SQSTM1 in different applications.
Automatically generated - may contain errors
Lab products found in correlation
Rabbit anti calponin, by Abcam (4 mentions)
Mouse anti ubiquitin, by Cell Signaling Technology (4 mentions)
Rabbit anti cdk1, by Cell Signaling Technology (4 mentions)
Mouse anti vimentin, by Abcam (4 mentions)
Rabbit anti α sma, by Abcam (3 mentions)
Rabbit anti lc3b, by Cell Signaling Technology (3 mentions)
Pvdf membrane, by Merck Group (3 mentions)
Image pro plus 6, by Media Cybernetics (3 mentions)
Rabbit anti lc3b, by Merck Group (2 mentions)
Alexa fluor 488 donkey anti rabbit igg, by Thermo Fisher Scientific (2 mentions)
Rabbit anti beclin1, by Abcam (2 mentions)
Rabbit anti gapdh, by Abcam (2 mentions)
Omat x ray film, by Kodak (2 mentions)
Goat anti β actin, by Santa Cruz Biotechnology (2 mentions)
Rabbit anti α smooth muscle actin sma, by Abcam (2 mentions)
Fluoview fv1000, by Olympus (2 mentions)
Rabbit anti lc3a b, by Cell Signaling Technology (2 mentions)
Rabbit anti collagen 1, by Novus Biologicals (1 mentions)
Bca protein assay kit, by Beyotime (1 mentions)
Rabbit anti pdgfr β, by Abcam (1 mentions)
20 protocols using rabbit anti p62
1
Renal Protein Extraction and Western Blot
2
Immunofluorescence Analysis of Mouse Brain
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In all, 10- to 12-week-old mice (n = 7–8 per group) were perfused transcardially with 4% paraformaldehyde. The brains were then frozen, sectioned at 30-µm thick, and processed for immunofluorescence using standard procedures7 (link). The primary antibodies used for IHC were as follows: sheep anti-αMSH antibody (1:40,000, Millipore, cat #AB5087), rabbit anti-AgRP (1:1,000, Phoenix Pharmaceuticals, cat #H003-53), rabbit anti-LC3B (1:500, Cell Signaling, cat #2775), rabbit anti-p62 (1:1,000, Abcam, cat #ab64134), rabbit anti-GFP (1:1,000, Invitrogen, cat #A-6455), guinea pig anti-insulin (1:500, Abcam, cat #ab7842), and rabbit anti-vesicular acetylcholine transporter (VAChT, 1:500, Synaptic Systems, cat #139103). The primary antibodies were visualized with Alexa Fluor 568 donkey anti-sheep IgG (1:200, Invitrogen, cat #A21099), Alexa Fluor 488 donkey anti-rabbit IgG (1:200, Invitrogen, cat #A21206), Alexa Fluor 488 donkey anti-mouse IgG (1:200, Invitrogen, cat #A21202), Alexa Fluor 568 donkey anti-rabbit IgG (1:200, Invitrogen, cat #A10042), or Alexa Fluor 488 goat anti-guinea pig IgG (1:200, Invitrogen, cat #A11073). The sections were counterstained using bis-benzamide (1:10,000, Invitrogen, cat #H3569) to visualize cell nuclei.
3
Quantifying Autophagy and Apoptosis Markers
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To determine APN, LC-3, Beclin 1, Akt/P-Akt, P62, C-caspase-3/P-caspase-3 levels, proteins were extracted from the cells by suspension in radioimmunoprecipitation assay (RIPA) buffer. Samples were centrifuged at 12,000 rpm at 4°C for 30 min, and the supernatants were recovered for analysis. The protein concentrations were determined using the Bradford protein method and the bicinchoninic acid (BCA) protein assay kit (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany). Protein (40 µg) was electrophoresed on a pre-cast bis-Tris polyacrylamide gel (12%), and then transferred onto a polyvinylidene difluoride (PVDF) membrane. Membranes were blotted with rabbit anti-APN (1:1,000), rabbit anti-LC3B (1:500), rabbit anti-beclin 1 (1:1,000), rabbit anti-Akt (1:1,000), rabbit anti-p-Akt (1:1,000), rabbit anti-P62 (1:1,000), rabbit anti-Caspase-3 (1:1,000) (all from Abcam, Cambridge, MA, USA) and mouse anti-actin (1:1,000; ProteinTech Group, Inc., Chicago, IL, USA), followed by horseradish peroxidase (HRP)-conjugated secondary antibodies (1:5,000; ZsBio, Beijing, China). Immunoblots were visualized using enhanced chemiluminescence (LAS-4000).
4
Autophagy Marker Immunoblotting Protocol
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The specific method is as mentioned before [28 (link)]. In this study, primary antibodies included rabbit anti-LC3B (1:1000; Sigma-Aldrich L7543), rabbit anti-P62 (1:1000; Abcam ab205719), mouse anti-β-Actin (1:1000; Sigma-Aldrich A5316), rabbit anti-Sidt2 (1:1000; Invitrogen PA5-69064), rabbit anti-Atg5 (1:1000; Cell Signaling Technology 9980S), rabbit anti-Atg7 (1:1000; Cell Signaling Technology 8558S), rabbit anti-Atg12 (1:1000; Cell Signaling Technology 2011S), mouse anti-LAMP1 (1:1000; Santa Cruz Biotechnology H4A3), rabbit anti-cathepsin B (1:1000; Cell Signaling Technology 31718S).
5
Protein Expression Analysis in Tissues
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Total protein of tissues and SH-SY5Y cell were collected with lysis buffer, and the concentrations of proteins were measured. Aliquot proteins were separated on 10% SDS-PAGE and electroblotted onto polyvinylidene difluoride membranes. The membrane was blocked with 5% nonfat dried milk for 2 h, subsequently, incubated with primary antibody overnight at 4°C. Related antibodies were as follows: rabbit anti-GAPDH (1 : 10000, Abcam), rabbit anti-PHD2 (1 : 500, Novusbio), rabbit anti-p62 (1 : 2000, Abcam), rabbit anti-Beclin-1 (1 : 1000, CST), rabbit anti-LC3 (1 : 1000, CST), and horseradish peroxidase (HRP) (1 : 10000, Aspen).
6
Western Blot Analysis of Autophagy and Inflammation Markers
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Total protein was extracted from tissues and NSCLC cells using ProteoPrep Sample Extraction kit (Sigma-Aldrich; Merck KGaA). The QuantiPro™ BCA Assay kit (cat. no. QPBCA; Sigma-Aldrich; Merck KGaA) was used to measure protein concentration. Protein samples (30 µg) were separated via SDS-PAGE (10% gel; Bio-Rad Laboratories, Inc.) and transferred to PVDF membranes. The membranes were then blocked with 5% non-fat milk in TBS with 0.05% Tween-20 for 1 h at room temperature. After the membranes were incubated with primary antibodies at 4˚C overnight, secondary antibodies were added and incubated with the membranes at room temperature for 1 h. The bands were visualized with an ECL Western Blotting substrate kit (cat. no. K820; BioVision, Inc.) and the protein expression was quantified using ImageJ v1.8.0 software (National Institutes of Health) with GAPDH as the loading control. The primary antibodies used included: Mouse anti-IL-1β (1:1,000; cat. no. 12242; Cell Signaling Technology, Inc.), rabbit anti-LC3B (1:1,000; cat. no. ab51520; Abcam), rabbit anti-P62 (1:10,000; cat. no. ab109012; Abcam) and rabbit anti-GAPDH (1:2,500; cat. no. ab9485; Abcam). The corresponding secondary antibodies included: Anti-mouse IgG HRP-conjugated antibody (1;2,000; cat. no. 7076; Cell Signaling Technology, Inc.) and goat anti-rabbit IgG HRP-conjugated antibody (1:20,000; cat. no. ab205718; Abcam).
7
Western Blot Analysis of Vascular Markers
8
Immunocytochemistry Analysis of Cultured CAMs
9
Muscle Protein Expression Analysis
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The muscles were homogenized in Tris-EDTA buffer with a cocktail of protease inhibitors and 1 mM phenylmethanesulfonyl fluoride. Proteins were subjected to SDS-PAGE, transferred onto polyvinylidene difluoride membranes (Millipore, USA), and probed with mouse anti-MHC (1 : 1,000) (MF-20, Developmental Studies, Hybridoma Bank, University of Iowa, USA), mouse anti-tubulin (1 : 5,000), mouse anti-GAPDH (1 : 5,000), rabbit anti-Bax (1 : 500), rabbit anti-Bcl2 (1 : 500), rabbit anti-caspase-3 (1 : 500), rabbit anti-Ub proteins (1 : 500) (Santa Cruz Biotechnology, USA), rabbit anti-LC3B (1 : 1,500) (Cell Signaling, USA), and rabbit anti-p62 (1 : 1,500) (Abcam, USA). All immunoreactions were visualized by enhanced chemiluminescence (Thermo Scientific, USA). Images were acquired using Fotodyne FOTO/Analyst Luminary Workstation Systems (Fotodyne, Inc., USA).
10
Immunocytochemistry Analysis of Cultured CAMs
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Cultured CAMs were grown on glass coverslips, stimulated or left unstimulated and fixed in 4% paraformaldehyde in phosphate-buffer saline (PFA/PBS) for 15 min. After permeabilization with 0.1% Triton X-100/PBS, the cells were rinsed with PBS and incubated overnight at 4°C with indicated primary antibodies: mouse anti-vimentin (1:200, Abcam), rabbit anti-α-SMA (1:200, Abcam), rabbit anti-calponin (1:200, Abcam), rabbit anti-p62 (1:200, Abcam), mouse anti-ubiquitin (1:200, Cell Signaling) and rabbit anti-CDK1 (1:200, Cell Signaling). The slides were extensively washed and incubated with Alexa-488- or Alexa-555-labeled secondary antibodies for 1 h at room temperature. The slides were mounted and subjected to examinations by using sequentially scanning on a laser scanning confocal microscope (Fluoview FV1000, Olympus, Japan), with photos being taken, and the co-localization analyzed by the Image Pro Plus 6.0 software (Media Cybernetics, Bethesda, MD, USA). The summarized co-localization efficiency data was expressed as Pearson correlation coefficient (PCC) as described previously [16 (link)].
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