For immunofluorescence analysis, NPCs were plated on chamber slides (Lab-Tek) or differentiated into neurons on poly-L-ornithine/laminin-coated glass-bottom culture dishes (MatTek). Cells were fixed in 4% (vol/vol) paraformaldehyde for 15 min followed by blocking in phosphate-buffered saline (PBS) containing 8% fetal bovine serum (vol/vol) for 1 h. The primary antibodies were prepared in PBS solution with 2 mg/ml saponin and incubated for 2 h at room temperature or overnight at 4°C. The cells were then washed in PBS and incubated with the corresponding fluorochrome-conjugated secondary antibodies for 1 h. Vectashield mounting medium plus DAPI (Vector Laboratories) was applied to label the nuclei. The following primary antibodies were used: anti-mTOR (Cell Signaling Technology, 2972) 1:100; anti-phospho-mTOR-Ser2448 (Cell Signaling Technology, 5536) 1:100; anti-S6 Ribosomal Protein (Cell Signaling Technology, 2217) 1: 200; anti-phosphoS6-Ser235/236 (Cell Signaling Technology, 2211) 1:100; anti-phospho-4EBP1-Thr37/46 (Cell Signaling Technology, 2855) 1:100; anti-LAMP1 (Developmental Studies Hybridoma Bank, H4A3) 1:200; anti-p62 (BD Biosciences, 610832) 1:100; anti-Tuj1 (Neuromics, MO15013) 1:200; anti-TFEB (MyBioSource, MBS855552) 1:50. The secondary antibodies used were Alexa fluor 488- or 594-conjugated mouse or rabbit (Life Technologies), both at a 1:200 or 1:400 dilution.
Anti lamp1
Manufactured by Developmental Studies Hybridoma Bank
Sourced in United States
Anti-LAMP1 is a laboratory reagent that functions as a marker for the lysosome-associated membrane protein 1 (LAMP1). LAMP1 is a widely expressed glycoprotein found on the lysosomal membrane of eukaryotic cells. The Anti-LAMP1 product can be used to detect and study the localization of LAMP1 in various cellular and experimental contexts.
Automatically generated - may contain errors
Lab products found in correlation
Anti p62, by Cell Signaling Technology (2 mentions)
Anti galectin 3, by Santa Cruz Biotechnology (2 mentions)
Annexin 5 alexa fluor 488 ready flow conjugate, by Thermo Fisher Scientific (2 mentions)
Anti alix, by Santa Cruz Biotechnology (2 mentions)
Anti c albicans, by GeneTex (2 mentions)
Anti goat secondary antibodies conjugated with alexa fluor 488 and horseradish peroxidase hrp, by Thermo Fisher Scientific (2 mentions)
Llome l7393, by Merck Group (2 mentions)
3 ma m9281 500mg, by Merck Group (2 mentions)
Anti lc3, by Thermo Fisher Scientific (2 mentions)
Alexa fluor 647, by Thermo Fisher Scientific (2 mentions)
Anti beclin1, by Merck Group (2 mentions)
Anti tsg101, by Abcam (2 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (2 mentions)
Anti s6 ribosomal protein, by Cell Signaling Technology (1 mentions)
Anti phospho 4e bp1 thr37 46, by Cell Signaling Technology (1 mentions)
Anti phospho s6 ser235 236, by Cell Signaling Technology (1 mentions)
Anti phospho mtor ser2448, by Cell Signaling Technology (1 mentions)
Anti mtor, by Cell Signaling Technology (1 mentions)
Vectashield plus dapi mounting medium, by Vector Laboratories (1 mentions)
Anti tuj1, by Neuromics (1 mentions)
11 protocols using anti lamp1
1
Immunofluorescence Analysis of mTOR Pathway
2
Immunoblotting Analysis of Candida Infection
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The primary antibodies are as follows: anti-C. albicans (catalogue #GTX40096, Genetex), anti-LAMP1 (catalogue #1D4B, Developmental Studies Hybridoma Bank), anti-Alix (catalogue #sc-53540 Santa Cruz Biotechnology), anti-Tsg101 (catalogue #AB83 Abcam), anti-Galectin3 (catalogue #sc-32790 Santa Cruz Biotechnology), anti-LC3 (catalogue #PA1-16930 Thermo Fisher Scientific), anti-p62 (catalogue #51145 Cell Signalling), anti-Beclin1 (catalogue #A-00023 Sigma), and anti-Cx43 (catalogue #AB0016–500 SICGEN). Anti-goat secondary antibodies conjugated with Alexa Fluor 488 and horseradish peroxidase (HRP) were purchased from Invitrogen and Life Technologies Jackson, respectively. Anti-mouse secondary antibodies conjugated with Alexa Fluor 568, Alexa Fluor 647 and HRP were purchased from Invitrogen and BioRad, respectively. Anti-rabbit secondary antibodies conjugated with Alexa Fluor 488, Alexa Fluor 647, and HRP were purchased from Invitrogen and BioRad, respectively. Anti-rat secondary antibodies conjugated with Alexa Fluor 594 and HRP were purchased from Invitrogen. Phalloidin was obtained from Sigma-Aldrich/Merck. LLOMe (L7393) and 3-MA (M9281-500MG) were purchased from Sigma-Aldrich/Merck. BAPTA-AM was obtained from Calbiochem (196419). Annexin V Alexa Fluor 488 Ready Flow Conjugate was purchased from Thermo Fisher Scientific (R37174).
3
Immunoblotting Analysis of Candida Infection
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The primary antibodies are as follows: anti-C. albicans (catalogue #GTX40096, Genetex), anti-LAMP1 (catalogue #1D4B, Developmental Studies Hybridoma Bank), anti-Alix (catalogue #sc-53540 Santa Cruz Biotechnology), anti-Tsg101 (catalogue #AB83 Abcam), anti-Galectin3 (catalogue #sc-32790 Santa Cruz Biotechnology), anti-LC3 (catalogue #PA1-16930 Thermo Fisher Scientific), anti-p62 (catalogue #51145 Cell Signalling), anti-Beclin1 (catalogue #A-00023 Sigma), and anti-Cx43 (catalogue #AB0016–500 SICGEN). Anti-goat secondary antibodies conjugated with Alexa Fluor 488 and horseradish peroxidase (HRP) were purchased from Invitrogen and Life Technologies Jackson, respectively. Anti-mouse secondary antibodies conjugated with Alexa Fluor 568, Alexa Fluor 647 and HRP were purchased from Invitrogen and BioRad, respectively. Anti-rabbit secondary antibodies conjugated with Alexa Fluor 488, Alexa Fluor 647, and HRP were purchased from Invitrogen and BioRad, respectively. Anti-rat secondary antibodies conjugated with Alexa Fluor 594 and HRP were purchased from Invitrogen. Phalloidin was obtained from Sigma-Aldrich/Merck. LLOMe (L7393) and 3-MA (M9281-500MG) were purchased from Sigma-Aldrich/Merck. BAPTA-AM was obtained from Calbiochem (196419). Annexin V Alexa Fluor 488 Ready Flow Conjugate was purchased from Thermo Fisher Scientific (R37174).
4
Histopathological Analysis of Toxoplasma Infection
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Four 5 μm sections from different areas of the brain and eye were stained by periodic acid Schiff hematoxylin (PASH) or hematoxylin and eosin stain respectively. Histopathologic changes (Brain: microglial nodules, perivascular and diffuse inflammation; Retina: disruption of architecture, perivascular and vitreal inflammation) were scored from 0 to 4 similar to previously described criteria36 (link),37 (link). Sections were also incubated with anti-T. gondii Ab (BioGenex) and Tomato lectin-DyLight 488 (Vector laboratories) that efficiently labels neural endothelial cells38 (link) (at 0.5 μg/ml it stains neural endothelial cells rather than microglia) or anti-CD31 Ab (Elabscience). Coronal sections at the septo-diencephalic region (level of the thalamus) were examined at X400. The numbers of clusters of T. gondii parasites within tomato lectin+ elongated structures (endothelial cells) were counted per whole coronal section. Brain sections were also stained with anti-LC3 (Abgent) or anti-LAMP-1 (Developmental Studies Hybridoma Bank) Abs. Accumulation of LC3 or LAMP-1 around T. gondii located in endothelial cells was defined as the presence of a ring-like structure that surrounds the parasite21 (link),22 (link). Slides were analyzed using Leica DMI 6000 B automated microscope equipped for epifluorescence microscopy.
5
Antibodies Used in ApoL9 Study
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The following antibodies were used in this study: anti-V5 (mouse monoclonal, R960-25, Thermo Fisher Scientific), anti-FLAG (mouse monoclonal, F1804, clone M2, Sigma-Aldrich), anti-LC3B (#2775, rabbit polyclonal, CST), anti-β-actin (A3854, rabbit polyclonal, HRP-conjugate, Sigma-Aldrich), anti-V5 agarose affinity gel (A7345, monoclonal clone V5-10, Sigma-Aldrich), anti-eIF3η (sc-16377 (N20), goat polyclonal, Santa Cruz Biotechnology, Inc.), anti-GFP (sc-9996, mouse monoclonal clone B2, Santa Cruz), anti-SQSTM1 (Ab56416, mouse monoclonal, Abcam), anti-V5 (V8137, rabbit polyclonal, Sigma-Aldrich), anti-LAMP1 (clone 1D4B, rat monoclonal, Developmental Studies Hybridoma Bank). Rabbit polyclonal anti-MBP-ApoL9 antibody has been described and validated earlier in Arvind and Rangarajan (2016) (link). Also it should be noted that in the bulk of experiments in the paper, ApoL9-V5 was detected in immunostaining and immunoblotting by anti-V5 (mouse) antibody, unless specifically mentioned otherwise.
6
Quantifying Autophagosome-Lysosome Colocalization
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HCT 116p53−/− and uL3ΔHCT 116p53−/− cells seeded on slides were fixed for 10 min with Methanol (Sigma-Aldrich, St. Louis, MO, USA), followed by incubation with blocking–permeabilization solution: 0.5% BSA, 0.1% saponin, and NH4Cl 50 mmol/L in phosphate buffered saline for 30 min. Primary antibodies anti-LC3B (Novus Biologicals, Centennial, CO, USA) and anti-LAMP1 (Developmental Studies Hybridoma Bank) were diluted in blocking–permeabilization solution and added to the cells for 1 h. Then, secondary antibodies were incubated for 45 min. Lastly, cells were counterstained with Hoechst (Thermo Fisher Scientific, Uppsala, Sweden). Samples were examined under a confocal microscope (Zeiss LSM 700; Carl Zeiss AG, Jena, Germany) equipped with 40x and 63× 1.4 NA oil objective. Colocalization between specific markers was quantified using Zeiss Zen 2012 software.
7
Immunostaining of Infected Cells
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Infected HeLa CCL2 and THP-1 cells were stained at day 3 post-infection as previously described [20 (link)], using anti-LAMP-1 (Developmental Studies Hybridoma Bank) and anti-Coxiella antibodies (Roy Laboratory, Yale University).
8
Autophagy Dynamics in Toxoplasma Infection
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Mammalian cells were infected with RH‐RFP T. gondii tachyzoites and fixed with 4% paraformaldehyde at 6 or 8 hr. LC3 and LAMP‐1 accumulation around the parasite were detected with anti‐LC3 (MBL international) and anti‐LAMP‐1 (Developmental Studies Hybridoma Bank, University of Iowa) antibodies and a secondary antibody conjugated to Alexa Fluor 488 (Jackson Immuno‐Research Laboratories). Accumulation of LC3 or LAMP‐1 around T. gondii was defined as the presence of a ring‐like structure that surrounds the parasite (Andrade et al., 2006 ; Van Grol et al., 2013 ). At least 50 cells per well (duplicate or triplicate wells per group per experiment) were counted manually. Slides were analysed using an Olympus FV1200 IX‐83 confocal microscope equipped with an UPlanSApa 60× objective, N.A. 1.35 oil. Images were acquired using FLUOVIEW v4.2b. Z‐stack images were deconvolved using MetaMorph v7.8.1.0. Images were processed in Photoshop CC 19.1.1. using similar linear adjustments for all samples.
9
Antibody Characterization for Vesicle Trafficking
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TAT1 anti-tubulin was a gift from Keith Gull (University of Oxford). The following commercial antibodies were used. Mouse: anti-LITAF (Santa Cruz); anti-transferrin receptor (Zymed); anti-Strep-Tag (Novagen); anti-CD63 (Millipore); anti-EEA1 (early endosome antigen 1; BD Biosciences); anti-LAMP1 (lysosome-associated membrane protein 1; Developmental Studies Hybridoma Bank, University of Iowa); anti-OPG2 (opsin tag containing two glycosylation sites) was described previously [29 (link)]. Rabbit: anti-TorsinA was a gift from Lisa Swanton (University of Manchester); anti-V5 (Abcam); anti-EEA1, anti-LAMP1 (Cell Signaling). Sheep: anti-GFP was an in-house antibody generated against GST-GFP. Fluorescent secondary antibodies for IF and for Licor immunoblotting were from Jackson ImmunoResearch Laboratories (PA, USA).
10
Quantifying Autophagy around Toxoplasma
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Endothelial cells challenged with RFP T. gondii were incubated with LC3 antibody (MBL International), or anti-LAMP-1 (Developmental Studies Hybridoma Bank) followed by secondary antibodies. Accumulation of LC3 or LAMP-1 around T. gondii was examined. At least 50 cells per well (duplicate or triplicate wells per group per experiment) were counted manually.
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