Automated immunostaining was carried out using Ventana BenchMarkXT platform (Ventana). The following antibodies were used; anti-vimentin and anti-cytokeratin (Ventana), rabbit anti-LC3A and rabbit anti-LC3B. Immunofluorescence was performed as described previously40 (link). For immunofluorescence, the following primary antibodies were used at the indicated concentrations: rabbit anti-LC3A (1:50) (Cat # 4599, Cell Signaling Technology), rabbit anti-LC3B (1:50) (Cat # 3868, Cell Signaling Technology), rabbit anti-vimentin (1:50) (Cat # 5741, Cell Signaling Technology), mouse anti-vimentin (1:100) (Cat # ab8978, Abcam), mouse anti-LAMP2 (1:50) (Cat # sc-18822, Santa Cruz Biotechnology), and mouse anti-LC3B (1:50) (Cat # sc-271625, Santa Cruz Biotechnology). Bound antibodies were visualized using Alexa Fluor 555 or Alexa Fluor 488 secondary antibodies (1:500) (Cell Signaling Technology). Cells were then counterstained using 4, 6-diamidinophenylindole (DAPI). Images were acquired using LSM 710 confocal scanning laser microscope (Carl Zeiss).
Rabbit anti lc3b
Manufactured by Cell Signaling Technology
Sourced in United States
Rabbit anti-LC3B is a primary antibody that specifically recognizes the microtubule-associated protein 1 light chain 3B (LC3B). LC3B is a widely used marker of autophagy and plays a crucial role in the formation of autophagosomes. This antibody can be used for the detection and analysis of LC3B in various experimental systems.
Automatically generated - may contain errors
Lab products found in correlation
Rabbit anti beclin 1, by Cell Signaling Technology (6 mentions)
Rabbit anti β actin, by Cell Signaling Technology (4 mentions)
Rabbit anti vimentin, by Cell Signaling Technology (3 mentions)
Rabbit anti p62, by Abcam (3 mentions)
Rabbit anti beclin1 antibody, by Cell Signaling Technology (3 mentions)
Rabbit anti sqstm1 p62, by Cell Signaling Technology (3 mentions)
Anti β actin, by Merck Group (3 mentions)
Bca assay, by Thermo Fisher Scientific (3 mentions)
Pvdf membrane, by Merck Group (3 mentions)
Rabbit anti lc3a, by Cell Signaling Technology (2 mentions)
Mouse anti lamp2, by Santa Cruz Biotechnology (2 mentions)
Mouse anti flag, by Merck Group (2 mentions)
3 methyladenine 3 ma, by Selleck Chemicals (2 mentions)
Mouse anti β actin ac 74, by Merck Group (2 mentions)
A5316, by Merck Group (2 mentions)
Mouse anti n cadherin, by BD (2 mentions)
Mouse anti p62 sqstm1 d 3, by Cell Signaling Technology (2 mentions)
Mouse anti cd44 clone 156 c11, by Cell Signaling Technology (2 mentions)
Mouse anti e cadherin, by BD (2 mentions)
Mg132, by Merck Group (2 mentions)
62 protocols using rabbit anti lc3b
1
Automated Immunostaining with Confocal Imaging
2
Evaluating Genistein and BPA Effects
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Genistein (purity>98%) was obtained from Fuzhou Rimian Inc. (Fuzhou, FuJian, China); and Bisphenol A (BPA), (CAS No.80-05-7, (CH3)2C(C6H4OH)2, ≥99% purity) was obtained from Sigma Chemical Company (St. Louis, Missouri). Assay kits for serum total cholesterol (TC), and triglycerides (TG) were purchased from BIOSINO Biotechnology and Science Inc. (Beijing, China). TRIZOL was obtained from Invitrogen Inc. (Carlsbad, CA, USA) and Real time quantitative PCR kit was purchased from TAKARA Bio Inc. (Otsu, Shiga, Japan). Insulin antibody was obtained from Merck Millipore (Billerica, MA, USA). Rabbit anti-LC3B was purchased by Cell Signaling Technology (Billerica, MA, USA), PPARα and PPARγ antibody were obtained from Abcam (Cambridge, UK). All other chemicals were of the highest grade commercially available.
3
Western Blot Analysis of Autophagy Markers
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Protein from cell lines was isolated with M-PER Mammalian Protein Extraction Reagent (78501, Thermo Scientific, MA, USA). Protein from prostate cancer tissue was extracted with Complete Lysis-M (13354520, Roche, Mannheim, Germany). Protein concentration was determined by the BCA method (23225, Beyotime, Shanghai, China). Proteins were separated using 6-12% SDS-PAGE and transferred onto nitrocellulose membrane (10401396, GE healthcare, OH, USA). The membranes were blocked by blocking buffer (927-40000, Odyssey, MA, USA) for 1 hour and then incubated in the primary antibodies at 4°C overnight. After washing with TBST, the membranes were incubated with secondary antibodies at room temperature for 1 hour. Protein bands were visualized by infrared imaging system (Odyssey) and quantified with Odyssey application software. Rabbit anti-LC3B, SQSTM1, ATG7, MTOR, p-P70S6k, and cleaved CASP3 antibodies were purchased from Cell Signaling Technology, MA, USA (2775, 5114, 8558, 2983, 9208, 9664). Anti-GAPDH antibody was purchased from Abcam, Cambridge, UK (9485).
4
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.
5
Antibody Validation for Protein Analysis
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The following primary antibodies were used in our study: mouse anti-CValpha (Abcam, 14748; 1:5000), rabbit anti-Hsp60 (Abcam, 46798; 1:500), rabbit anti-ROCK2 (Abcam, 125025; 1:5000), mouse anti-actin (Abcam, 8226; 1:1000), mouse anti-HK2 (Abcam, 3740910; 1:1000), mouse anti-Mfn2 (Abcam, 56889; 1:1000), rabbit anti-TOM20 (Santa Cruz, 191883; 1:1000), mouse anti-VDAC1 (Abcam, 14734; 1:1000), mouse anti-TOM70 (Santa Cruz, 390545; 1:1000), mouse anti-UQCRC2 (Abcam, 14745, 1:1000), rabbit anti-COXIV (Novus, NB110-39115; 1:500) and mouse anti-Flag (Sigma, F1804; 1:1000). Rabbit and mouse horseradish peroxidase-conjugated secondary antibodies (Jackson Immunoresearch; 1:5000) were used for secondary incubation.
The following primary antibodies were used in our study for immunofluorescence: mousen anti-CValpha (Abcam, 14748; 1:500), rabbit anti-Hsp60 (Abcam, 46798; 1:500), rabbit anti-LC3B (Cell Signaling, 2775) and mouse anti-Flag (Sigma, F1804; 1:1000). All antibodies were diluted in 1% goat serum.
The following primary antibodies were used in our study for immunofluorescence: mousen anti-CValpha (Abcam, 14748; 1:500), rabbit anti-Hsp60 (Abcam, 46798; 1:500), rabbit anti-LC3B (Cell Signaling, 2775) and mouse anti-Flag (Sigma, F1804; 1:1000). All antibodies were diluted in 1% goat serum.
6
Investigating Autophagy Regulation via PHB2
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The following antibodies were used: rabbit anti-LC3B (Cell Signaling Technology, Boston, MA, USA, 3868), rabbit anti-PHB2 (Cell Signaling Technology, 14085), rabbit anti-SQSTM1/p62 (ABclonal Technology, Wuhan, China, A77580), normal rabbit IgG (ABclonal Technology, AC005), mouse anti-PHB2 (Proteintech, Wuhan, China, 66424-1-Ig), mouse anti-β-actin (Proteintech, 66009-1-Ig), rabbit anti-flag (Proteintech, 20543-1-AP), and rabbit anti-EV-A71 VP1 (Genetex, Irvine, CA, USA, GTX132339).
The following reagents were used: 3-methyladenine (3-MA; Selleck, Shanghai, China, S2767), rapamycin (Selleck, S1039), nontargeting siRNA (SiNC) and siRNA targeting PHB2 (SiPHB2) and ATP6AP2 (SiATP6AP2) (RiboBio, Guangzhou, China), Lipofectamine 3000 Reagent (Invitrogen, Carlsbad, CA, USA, L3000015), Lipofectamine RNAiMAX Reagent (Invitrogen, 13778-150), and primers for PCR and real-time quantitative PCR (RT-qPCR) (Invitrogen).
The following reagents were used: 3-methyladenine (3-MA; Selleck, Shanghai, China, S2767), rapamycin (Selleck, S1039), nontargeting siRNA (SiNC) and siRNA targeting PHB2 (SiPHB2) and ATP6AP2 (SiATP6AP2) (RiboBio, Guangzhou, China), Lipofectamine 3000 Reagent (Invitrogen, Carlsbad, CA, USA, L3000015), Lipofectamine RNAiMAX Reagent (Invitrogen, 13778-150), and primers for PCR and real-time quantitative PCR (RT-qPCR) (Invitrogen).
7
Comprehensive Antibody Panel for Autophagy and UPR Analysis
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Rabbit anti-LC3B (#3868), rabbit anti-ATG5 (#12994), rabbit anti-ATG7 (#8558), rabbit anti-Beclin-1 (#3495), rabbit anti-ATG16L1 (#8089), rabbit anti-BIP (#3177), rabbit anti-ATF6 (#65880), rabbit anti-ATF4(#11815), rabbit anti-XBP1 (#12782), rabbit anti-PERK (#5683), rabbit anti-IRE1 (#3294), rabbit anti-p-eif2α (#3398), rabbit anti-FIP200 (#12436), and mouse anti-CHOP (#2895) antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA) (dilution concentration 1:1000). Rabbit anti-p-IRE1 (PA1-16927) was purchased from Invitrogen (Waltham, MA, USA) (dilution concentration 1:1000). Mouse anti-p62 (18420-1-AP), mouse anti-HA-tag (66006-2-Ig), and mouse anti-β-actin (66009-1-Ig) were purchased from Proteintech (Wuhan, China) (dilution concentration 1:5000). HRP-conjugated goat anti-mouse (G1214) and goat anti-rabbit (G1213) were purchased from Servicebio (Wuhan City, China) (dilution concentration 2:5000). Alexa Fluor 568 goat anti-mouse IgG, IgM (H + L) (A-11004) and Alexa Fluor 647 goat anti-mouse-IgG (H + L) (A-21445) were purchased from Thermo Fisher (Waltham, MA, USA) (dilution concentration 1:500). Immunofluorescence antibodies were diluted in PBS. Immunoblot antibodies were diluted in TBST.
8
Comprehensive Western Blotting Approach
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Western blotting was performed as described previously18 (link), 22 (link) using the following primary antibodies: rabbit anti-LC3B (1:1000; 2775; Cell Signaling Technology), mouse anti-p62/SQSTM1 (D-3) (1:1000; sc-28359; Cell Signaling Technology), rabbit anti-Beclin1 antibody (1:1000; 3738; Cell Signaling Technology), mouse anti-E-cadherin (1:10000; 610182; BD Biosciences), mouse anti-N-cadherin (1:1000; 610921; BD Biosciences), and mouse anti-β-Actin (AC-74) (1:10000; A5316; Sigma-Aldrich). To detect CD44, we utilized mouse anti-CD44 clone 156-C11 (1:1000; 3570; Cell Signaling Technology) which recognizes an epitope in the amino-terminal extracellular region of CD4448 , allowing for detection of the standard isoform of CD44 as well as variant CD44 isoforms. CD44v6 expression was evaluated via mouse anti-CD44v6 clone 2F10 (1:500; BBA13 R&D Systems). Densitometry was performed with Image J (National Institutes of Health).
9
Immunofluorescence Assay for LC3B
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C2C12 cells were grown, transduced with adenovirus, and treated on glass coverslips. At the end of the experiment, cells were washed twice in ice-cold PBS-Ca+2/Mg+2, fixed in methanol, blocked for 1 h in 10% (v/v) goat serum in PBS, and incubated for 1 h with specific antibody rabbit anti-LC3B (1:200; Cell Signaling, Danvers, MA, USA). The bound antibodies were detected by incubating the cells for 1 h with 1:10,000 affinity-purified Alexa Fluor dye-conjugated goat anti-rabbit antibody (Thermo Scientific, Waltham, MA, USA). After rinsing, the cells were mounted with fluorescence mounting medium (Agilent Dako, Santa Clara, CA, USA) under a glass slide and viewed and photographed using the Motic BA310 fluorescence microscopy (Motic, Hong Kong, China).
10
Antibodies and Chemical Inhibitors Protocol
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The mouse polyclonal antibody against CP204L and rabbit polyclonal antibody against B646L were prepared in our laboratory. Polyclonal rabbit anti-SNX32 (catalog no. 25763-1-AP), rabbit anti-p62 (catalog no. 18420-1-AP), and rabbit anti-RAB1B (catalog no. 17824-1-AP) were purchased from Proteintech. Rabbit anti-LC3B (catalog no. 3868s) and mouse anti-Myc (catalog no. 2276S) were purchased from Cell Signaling Technology. Monoclonal mouse anti-HA (catalog no. H3663), mouse IgG polyclonal antibody (catalog no. 12-371), rabbit IgG polyclonal antibody (catalog no. 12-370), and mouse anti-Flag (catalog no. F1804) were purchased from Sigma-Aldrich. Mouse anti-β-actin was purchased from Santa Cruz (catalog no. sc-58673). Alexa Fluor 488-conjugated goat anti-mouse IgG (H+L) (catalog no. 4408s) and Alexa Fluor 594-conjugated goat anti-rabbit IgG (H+L) (catalog no. 8889s) antibodies were purchased from Cell Signaling Technology; IPKine goat anti-mouse IgG heavy chain secondary antibody, HRP labeling (elimination of light chain interference) (catalog no. A25112) and IPKine goat anti-mouse IgG light chain secondary antibody, HRP labeling (elimination of heavy chain interference) (catalog no. A25012) were purchased from Abbkine. Dimethyl sulfoxide, 3-MA (autophagosome formation inhibitor), MG132 (proteasome inhibitor), and NH4Cl (lysosome inhibitor) were purchased from Sigma-Aldrich.
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