HeLa-tTA cells (3.5 × 104) expressing FA or a traptamer were seeded on glass cover slips in 24-well plates, incubated overnight, and mock-infected or infected with wild-type HPV16-HcRed PsV at MOI of 150. At various h.p.i., cells were fixed for 15 min at room temperature with 4% paraformaldehyde, permeabilized with 1% saponin for 30 min, and incubated with 1:400 anti-FLAG mouse antibody (Sigma-Aldrich, F3165), 1:100 anti-EEA1 rabbit antibody [Cell Signaling Technology (CST), 2411], 1:75 anti-Rab7 rabbit antibody (CST, 9367), 1:300 anti-TGN46 rabbit antibody (Abcam, ab50595), or 1:100 anti-LAMP1 rabbit antibody (Abcam, ab24170) at 4°C overnight. Cells were then incubated at room temperature for 1 hour with 1:200 Alexa Fluor–conjugated secondary antibody (Life Technologies). The slides were mounted in mounting solution with 4′,6-diamidino-2-phenylindole (DAPI), and images were captured using a Leica SP5 confocal microscope. To quantify colocalization, Pearson’s correlation coefficient between channels was obtained in ImageJ by using the coloc2 plugin (NIH; https://imagej.net/Coloc_2 ).
Rabbit anti lamp1 antibody
Manufactured by Abcam
Sourced in United States
The Rabbit anti-LAMP1 antibody is a primary antibody that specifically binds to the lysosome-associated membrane protein 1 (LAMP1) in rabbit samples. LAMP1 is a membrane glycoprotein that is primarily found in the lysosomal membrane and is commonly used as a marker for lysosomes.
Automatically generated - may contain errors
Lab products found in correlation
Anti tgn46 rabbit antibody, by Abcam (2 mentions)
Mouse anti flag antibody, by Merck Group (1 mentions)
Alexa fluor conjugated secondary antibody, by Thermo Fisher Scientific (1 mentions)
Sp5 confocal microscope, by Leica (1 mentions)
Alexa fluor 488 imaging kit, by Thermo Fisher Scientific (1 mentions)
5 ethynyl 2 deoxyuridine (edu), by Thermo Fisher Scientific (1 mentions)
Las x software, by Leica (1 mentions)
Alexa fluor 488 goat anti rabbit igg, by Thermo Fisher Scientific (1 mentions)
Mitosox red reagent, by Thermo Fisher Scientific (1 mentions)
Alexa flour 555 goat anti rabbit igg, by Thermo Fisher Scientific (1 mentions)
Lsm 880 confocal, by Zeiss (1 mentions)
Cy3 labeled goat anti rabbit igg h l, by Beyotime (1 mentions)
Sp8 confocal microscope, by Leica (1 mentions)
Paraformaldehyde (pfa), by Merck Group (1 mentions)
Triton x 100, by Merck Group (1 mentions)
Mouse anti gfap antibody, by Cell Signaling Technology (1 mentions)
Mouse anti β actin antibody, by Abcam (1 mentions)
Rabbit anti synaptophysin antibody, by Abcam (1 mentions)
Protein a g sepharose, by Santa Cruz Biotechnology (1 mentions)
Mouse anti glut4 antibody, by Santa Cruz Biotechnology (1 mentions)
6 protocols using rabbit anti lamp1 antibody
1
Investigating HPV Infection Dynamics in HeLa-tTA Cells
2
Visualizing HPV16 Pseudovirus Uptake
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To produce HPV16 PsV containing reporter plasmids substituted with EdU, 100 μM EdU (Thermo Fisher Scientific, C10337) was added to the 293TT cell growth medium at 6 hours after transfection of pSheLL16 L2 and the HcRed reporter plasmid. Cells were harvested and PsV was purified as described above. HeLa cells were mock-infected or infected at MOI of 150 with EdU-labeled HPV16 PsV. At various h.p.i., the cells were fixed in 4% paraformaldehyde and permeabilized with 1% saponin. The cells were incubated with the Click-iT reaction mixture prepared according to the protocol provided using the Alexa Fluor 488 Imaging Kit (Thermo Fisher Scientific, C10425). The cells were then incubated with antibodies recognizing cellular proteins [1:100 anti-EEA1 mouse antibody (BD Biosciences, #610457), 1:300 anti-TGN46 rabbit antibody (Abcam, ab50595), 1:100 anti-LAMP1 rabbit antibody (Abcam, ab24170), or 1:50 anti-PML mouse antibody (Santa Cruz Biotechnology, sc-966)] and the appropriate secondary antibodies provided by the manufacturer. Cells were imaged with a Leica SP8 gated STED 3X super-resolution microscope. The images were deconvolved by Huygens software (Scientific Volume Imaging) and then processed by LAS X software (Leica Microsystems).
3
Mitochondrial ROS and Autophagy Analysis
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BMDMs were incubated with MitoSOX Red reagent (Thermo Scientific) reagent (1 μM) to examine mitochondrial ROS generation which was visualized by fluorescence microscopy. Cells were also incubated with 10 nM MitoTracker Green (which is insensitive to ROS) to confirm the localization of MitoSOX Red to mitochondria. Immunofluorescence microscopy was carried out to visually identify p62 puncta and lysosomes and to determine co-localization of p62 and lysosomal-associated membrane protein 1 (LAMP1), which indicates autophagosome and lysosome fusion (i.e., activated autophagy). In brief, cells were fixed and permeabilized with cold methanol. Immunocytochemical staining of cells used rabbit anti-p62 monoclonal antibody (Cell Signaling, #23214) or rabbit anti-LAMP1 antibody (Abcam, #ab24210). Alexa Fluor 488 goat anti-rabbit IgG (Thermo Scientific) and Alexa Flour 555 goat anti-rabbit IgG (Thermo Scientific) secondary antibodies were used to detect p62 and LAMP1, respectively. Imaging was acquired via a confocal microscope (Zeiss LSM 880 Confocal with FAST Airyscan).
4
Immunofluorescence Imaging of ATP6V0D1 and LAMP1
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Cells were fixed with 4% (v/v) paraformaldehyde (Sigma‐Aldrich) and permeabilized with 0.3% (v/v) Triton X‐100 (Sigma‐Aldrich) for 3 min. Then cells were incubated with rabbit anti‐ATP6V0D1 primary antibody or rabbit anti‐LAMP1 antibody (a dilution of 1: 200, Abcam) at 4 °C overnight. Then cells were incubated with Cy3‐labeled goat anti‐rabbit IgG (H + L, a dilution of 1:500, Beyotime) at room temperature for 1 h. Nuclei were stained with DAPI for 2 min. The images of ATP6V0D1 were captured by a SP8 confocal microscope (Leica).
5
Immunoassay Antibody Validation Protocol
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In this study, IHC, ICC and Western blots assays utilized the following primary antibodies: Mouse anti-β-actin antibody (Abcam, #ab8226, Cambridge, UK), Mouse anti-GFAP antibody (Cell Signaling Technology, #3670S, Boston, MA, USA), Goat anti-PSD95 antibody (Abcam, #ab12093), Rabbit anti-synaptophysin antibody (Abcam, #ab32127), Rabbit anti-LAMP1 antibody (Abcam, #ab24170), Rabbit anti-MEGF10 antibody (Sigma-Aldrich, #ABC10, St. Louis, MO, USA).
6
Autophagy Regulation Mechanisms Elucidation
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Bafilomycin A1 (Baf A1, 20 μM) was purchased from Selleck (USA), and the classical autophagy inhibitor 3-methyladenine (3-MA, 1 mM) was purchased from Sigma-Aldrich (USA). Protein A/G-Sepharose (Santa Cruz, USA) was used in the immunoprecipitation assays. The antibodies used in this study included rabbit anti-MPR (cation independent) antibody (Abcam, USA), rabbit anti-α-Tubulin antibody (Proteintech, USA), rabbit anti-cathepsin B antibody (Santa Cruz, USA), mouse anti-cathepsin D antibody (Santa Cruz, USA), rabbit anti-Lamp1 antibody (Abcam, USA), rat anti-Lamp1 antibody (Abcam, USA), mouse anti-Golgin 160 antibody (Santa Cruz, USA), mouse anti-EEA1 antibody (Santa Cruz, USA), mouse anti-GLUT4 antibody (Santa Cruz, USA), mouse anti-TBC1D5 antibody (Santa Cruz, USA), mouse anti-VPS35 antibody (Santa Cruz, USA), mouse anti-VPS29 antibody (Santa Cruz, USA), mouse anti-SNX1 antibody (Santa Cruz, USA), mouse anti-SNX2 antibody (Santa Cruz, USA), mouse anti-SNX5 antibody (Santa Cruz, USA), mouse anti-SNX6 antibody (Santa Cruz, USA), mouse anti-Rab7 antibody (Abcam, USA), HRP-conjugated mouse anti-Actin antibody (Proteintech, USA), mouse anti-α-Tubulin antibody (Proteintech, USA), rabbit anti-ATG5 antibody (Cell Signaling Technology, USA) and rabbit anti-Dynactin p150glued antibody (Cell Signaling Technology, USA).
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