Confocal microscopy was performed as described previously.17 (link) After the appropriate treatments, the cells on coverslips were washed three times with fresh PBS, fixed with 4% paraformaldehyde for 15 min, permeabilized with 0.25% Triton X-100 (Sigma, St Louis, MO, USA) for 10 min, and incubated with LC3 antibodies (Cell Signaling Technology, Danvers, MA, USA, #4108) or LAMP1 antibodies (Cell Signaling Technology Cat# 3243, RRID:AB_2134478) for 2 h at room temperature. The cells were washed with PBS to remove excess primary antibodies and incubated with secondary antibodies for 1 h at room temperature. Nuclei were stained with DAPI for 1 min. After mounting, fluorescence images were acquired using a confocal laser-scanning microscope (Zeiss 780; Carl Zeiss GmbH, Oberkochen, Germany).
Lamp1 antibody
Manufactured by Cell Signaling Technology
Sourced in United States
The LAMP1 antibody is a laboratory reagent used to detect the presence and localization of the LAMP1 (Lysosome-Associated Membrane Protein 1) protein in biological samples. LAMP1 is a highly glycosylated type I membrane protein that is primarily found in the membrane of lysosomes, which are organelles responsible for the degradation and recycling of cellular components. The LAMP1 antibody can be used in various techniques, such as Western blotting, immunohistochemistry, and immunocytochemistry, to study the expression and distribution of LAMP1 in different cell types and tissues.
Automatically generated - may contain errors
Lab products found in correlation
Rpmi 1640 medium, by Thermo Fisher Scientific (2 mentions)
Fetal bovine serum (fbs), by Merck Group (2 mentions)
Lc3 antibody, by Cell Signaling Technology (1 mentions)
Triton x 100, by Merck Group (1 mentions)
Gentamycin, by Thermo Fisher Scientific (1 mentions)
Paraformaldehyde (pfa), by Santa Cruz Biotechnology (1 mentions)
Tlr4 pe, by BD (1 mentions)
Lsm 800, by Zeiss (1 mentions)
Lc3b antibody, by Cell Signaling Technology (1 mentions)
Nonidet p 40, by Merck Group (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions)
Revert total protein stain solution, by LI COR (1 mentions)
Gapdh antibody, by OriGene (1 mentions)
Odyssey blocking buffer, by LI COR (1 mentions)
Lysosome enrichment kit for tissues and cultured cells, by Thermo Fisher Scientific (1 mentions)
Bz x710, by Keyence (1 mentions)
8 protocols using lamp1 antibody
1
Confocal Microscopy of Autophagy Markers
2
TLR4 Endocytosis and Salmonella Infection
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To study TLR4 endocytosis, macrophages were stimulated with LPS (200 ng/ml) for indicated time. After treatment, cells were fixed by 3.7% Paraformaldehyde in PBS (Santa Cruz) for 20 minutes, permeablized by 0.5% Nonidet P40 (Sigma-Aldrich) and blocked in 1% BSA. Cells were subsequently stained with TLR4-PE (BD), EEA1-FITC (BD), and LAMP1 antibodies (Cell Signaling Technology). To assess Salmonella typhimurium infection, cells were infected with MOI 10 for one hour, then incubated with lysotracker Deep Red (0.1%, Thermo Fisher Scientific) and gentamycin (50 g/ml, Thermo Fisher Scientific) for one hour. Cells were then fixed and permeablized as described above, and stained with LC3B antibody (Cell Signaling Technology) and DAPI (Sigma Aldrich). Imaging was performed and analyzed using Zeiss LSM800 (Zeiss).
3
Characterizing anti-LY6H antibody internalization
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Example 7
Experiments were performed to characterize anti-LY6H antibody internalization in H446 cells. The following methods were used.
Methods
Tissue Culture and Cell Lines
Human leukemia cell line H446 was obtained from American Type Culture Collection (ATCC). The cells were maintained in RPMI-1640 medium (Invitrogen) with 10% fetal bovine serum (FBS) (Sigma).
Internalization Assay
Live H446 cells were incubated with 12G7 antibody for 30 minutes at 37° C. After cytospin, cells were fixed with 4% PFA and permeablized with 100% methanol, and stained with LAMP1 antibody (#9091, Cell Signaling Technology, Inc.).
Results
Antibody 12G7 was co-localized to lysozyme, marked by LAMP1 antibody (see
4
Immunoblot and Immunofluorescence Antibody Protocols
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All C9ORF72 antibodies are listed in Supplementary file 1 . SMCR8 antibody is from Abcam (ab202283). WDR41 antibody is from Abgent (AP10866B-EV). TOM20 antibody is from Santa Cruz Biotechnology, Inc (sc-11414). PDI antibody is from Abcam (ab2792). GAPDH antibody is from OriGene (TA802519). LAMP1 antibody used for immunoblot is from Cell Signaling Technology (#9091). LAMP1 antibody used for IF in RAW264.7 cells is from Thermo (PA1-654A). Peroxidase-conjugated goat anti-mouse and anti-rabbit are from Jackson ImmunoResearch Laboratories. Odyssey IRDye 800CW goat anti-mouse-HRP, the REVERT total protein stain solution and the Odyssey Blocking buffer (TBS) are from LI-COR Biosciences. Alexa Fluor 647-conjugated mouse and rabbit secondary antibodies are from Invitrogen.
5
Lysosomal Trafficking of Neurodegenerative Proteins
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To confirm trafficking of WT and mutant α-syn, TDP-43 and tau into the lysosomes of our SH-SY5Y cells, we performed a series of lysosome isolation experiments. SH-SY5Y cells were differentiated and treated with MG132 as previously described [37 (link), 38 (link)]. Cells were harvested on day 14 post-differentiation and the lysosomes of these cells isolated using a gradient-ultracentrifuge based technique (Lysosome Enrichment Kit for Tissues and Cultured Cells from Thermofisher, # 89,839). Successful isolation and enrichment of lysosomes were confirmed by Western Blot using a Lamp1 antibody (Cell Signaling #9091).
6
Anti-IL1RAP Antibody Internalization in EOL-1 Cells
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Example 5
Experiments were performed to characterize anti-IL1RAP antibody internalization in EOL-1 cells. The following methods were used.
Methods
Tissue Culture and Cell Lines
Human leukemia cell lines EOL1 was obtained from DSMZ. They were maintained in RPMI-1640 medium (Invitrogen) with 10% fetal bovine serum (FBS) (Sigma).
Internalization Assay
Live EOL1 cells were incubated with 44E5_15C5 antibody for 30 minutes at 37° C. After cytospin, cells were fixed with 4% PFA and permeablized with 100% methanol, and stained with LAMP1 antibody (#9091, Cell Signaling Technology, Inc.).
Results
Live EOL1 cells were incubated with 44E5_15C5 for 0.5 hours at 37° C. Cells were then fixed, permeablized, and co-stained with LAMP1 antibody. 44E5_15C5 is co-localized to lysozyme, marked by LAMP1 antibody (see
7
Immunofluorescent Labeling of LAMP-1 and Lipids
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Cells were fixed with 4% paraformaldehyde in PBS at room temperature for 15 minutes. Cells were then permeabilized with 0.1% Triton X-100 in PBS at room temperature for 5 min and blocked with 1% goat serum in PBS (blocking buffer) for 30 min. Cells were then incubated with LAMP-1 antibody (Cell signaling Technologies, #9091) in blocking buffer at room temperature for 1 hour, followed by incubation with secondary antibody (Invitrogen, #A-11008) in blocking buffer at room temperature for 30 min. Cells were then stained with 50 μg/ml filipin in PBS at room temperature for 2 hours. After filipin staining, cells were washed with PBS three times and mounted. Images were captured using an all-in-one fluorescence microscope (BZX-710; Keyence).
8
Internalization of Anti-ICAM1 Antibody in Myeloma
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