HeLa cells were plated in a 6-well plate (2 × 105 cells/well) and allowed to adhere overnight. Cells were infected with WT, Δstmp1 mutant, or Δstmp1 complement bacteria for 2 h, washed extensively with PBS, and scraped into 2 mL of 10% RPMI. Infected cells were replated onto coverslips in a 24-well plate (5 × 103 cells per well). At 6 dpi, cells were fixed with 2.5% paraformaldehyde (PFA) for 15 min, washed in PBS, and blocked/permeabilized in 1% BSA and 0.1% saponin in PBS for 20 min. Coverslips were stained with rabbit anti-LAMP1 (1:1,000; Abcam, Cambridge, UK) along with guinea pig anti-C. burnetii (1:2,500) for 1 h followed by Alexa Fluor secondary antibodies (1:1,000; Invitrogen, Carlsbad, CA) for 1 h. Following washing with PBS, coverslips were mounted with ProLong gold with DAPI and visualized on a Leica inverted DMI6000B microscope using a 63× oil immersion objective. Images were captured and processed identically, and the CCV area was measured using ImageJ software. At least 30 CCVs were measured per condition for each of three independent experiments.
Rabbit anti lamp1
Manufactured by Abcam
Sourced in United States
Rabbit anti-LAMP1 is a primary antibody that recognizes the lysosome-associated membrane protein 1 (LAMP1) in various species. LAMP1 is a transmembrane glycoprotein that is predominantly localized in the lysosomal membrane and plays a role in lysosomal biogenesis and function. This antibody can be used for the detection and localization of LAMP1 in various applications, such as Western blotting, immunohistochemistry, and immunocytochemistry.
Automatically generated - may contain errors
Lab products found in correlation
Alexa fluor secondary antibody, by Thermo Fisher Scientific (2 mentions)
Rabbit anti ha, by Abcam (2 mentions)
Dmi6000b inverted microscope, by Leica (1 mentions)
Mouse anti olig1, by Merck Group (1 mentions)
Rabbit anti ng2, by Abcam (1 mentions)
Prolong gold antifade reagent, by Thermo Fisher Scientific (1 mentions)
Mouse anti neun, by Merck Group (1 mentions)
Mouse anti β tubulin, by Merck Group (1 mentions)
Rabbit anti gfap, by Agilent Technologies (1 mentions)
Rabbit anti calbindin, by Merck Group (1 mentions)
Fluoromount g, by Southern Biotech (1 mentions)
Goat anti kim 1 antibody, by R&D Systems (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (1 mentions)
Anti giantin, by Abcam (1 mentions)
Rabbit anti asyn, by Abcam (1 mentions)
Triton x 100, by Merck Group (1 mentions)
Hoechst 33258, by Thermo Fisher Scientific (1 mentions)
Mouse anti asyn, by BD (1 mentions)
Anti lc3b, by Merck Group (1 mentions)
Rabbit anti cathepsin d, by Abcam (1 mentions)
20 protocols using rabbit anti lamp1
1
Quantifying Coxiella Replication in HeLa Cells
2
Immunofluorescent Staining of Neural Cells
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Cell cultures or brain slices were fixed in 4% paraformaldehyde in PBS, rinsed, permeabilized in 0.1% (for cells) or 3% (for slices) Triton X-100 in PBS, and then blocked with 5% normal goat serum (NGS) or normal donkey serum (NDS) in PBS containing 0.1% (for cells) or 0.3% (for slices) Triton X-100 for 1 h. Samples were then incubated with primary antibodies in blocking buffer, washed, and then probed with secondary antibodies (Alexa Fluor secondary antibodies, all from Life Technologies). After washing, the coverslips were mounted with ProLong Gold anti-fade reagent (Life Technologies). The brain slices were mounted with Fluoromount-G (Southern Biotech, Birmingham, AL), placed under the glass covers, and sealed. Primary antibodies used in the immunostaining were mouse anti-NeuN (Millipore), mouse anti-β-tubulin (Sigma), rabbit anti-GFAP (DAKO, Carpinteria, CA), mouse anti-Olig1(Millipore), mouse anti-O4 IgM (R&D Systems, Minneapolis, MN ), rabbit anti-LAMP1(Abcam, Cambridge, UK), rabbit anti-Calbindin (Millipore), mouse anti-C3d (a gift from Dr. Joshua Thurman, University of Colorado), rabbit anti-MAC complex (Abcam), and rabbit anti-NG2 and rabbit anti-Olig2 (both are gifts from Dr. Charles Stiles, Harvard University).
3
Immunofluorescence Analysis of Autophagy and Kidney Injury Markers
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Rabbit anti-LC3 and anti-Atg7 antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA, cat#12741 and #8540), guinea pig anti-p62 antibody was form PROGEN Biotech GmbH (Heidelberg, Germany, cat#GP62-C), mouse anti-Megalin antibody was from NOVUS (Littleton, CO, USA, cat#NB110-96417), goat anti-Kim-1 antibody was from R&D systems (Minneapolis, MN, USA, cat#AF1817), and rabbit anti-Lamp1 was from Abcam (Tokyo, Japan, cat# ab24170). Donkey Alexa 488- conjugated anti-goat IgG, anti-mouse IgG, anti-rabbit IgG, and Donkey Cy3- conjugated anti-guinea pig IgG antibodies (Jackson Immuno Research, Philadelphia, PA, USA, cat#705-545-003 and 706-165-148) 4′,6-Diamidino-2-Phenylindole (DAPI) was purchased from Thermo Fisher Scientific (Waltham, MA, USA, cat#D1306).
4
Immunofluorescence Staining of Transfected Cells
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Twenty-four or forty-eight hours after transfection, cells (HEK or H4) were washed with PBS and fixed with 4% paraformaldehyde (PFA) for 10 minutes at room temperature (RT), followed by a permeabilization step with 0.5% Triton X-100 (Sigma-Aldrich, St. Louis, MO, USA) for 20 minutes at RT. After blocking in 1.5% normal goat serum (PAA, Cölbe, Germany)/DPBS for 1 hour, cells were incubated with primary antibody. Primary antibodies used were: mouse anti-ASYN (1∶1000, BD Transduction Laboratory, New Jersey, USA) or rabbit anti-ASYN (1∶1000, Abcam, Boston, USA), rabbit anti-LAMP-1 (1∶1000, Abcam, Boston, USA), anti-Giantin (1∶1000, Abcam, Boston, USA) for 3 hours or overnight and secondary antibody (Alexa Fluor 488 donkey anti-mouse IgG and/or Alexa Fluor 555 goat anti rabbit IgG, (Life Technologies- Invitrogen, Carlsbad, CA, USA) for 2 hours at RT. Finally, cells were stained with Hoechst 33258 (Life Technologies- Invitrogen, Carlsbad, CA, USA) (1∶5000 in DPBS) for 5 minutes, and maintained in PBS for epifluorescence microscopy.
5
Evaluating Autophagy Markers in H. pylori Infection
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AGS cells (6 × 105) or GES-1 cells (4 × 105) were seeded on 6-well plates for 16 h and infected with H. pylori (MOI 100) for 6 h. The infected cells were washed five times with DPBS, and whole-cell lysates were prepared in radioimmunoprecipitation assay buffer (Sigma-Aldrich) with protease inhibitor cocktail (Calbiochem) and phosphatase inhibitor cocktail (Calbiochem). The samples were then analyzed by SDS-PAGE. The proteins were then transferred to a polyvinylidene difluoride membrane (GE Healthcare). Each membrane was blocked with 5% (w/v) dry milk in Tris-buffered saline (50 mM Tris, 150 mM NaCl, 1 mM CaCl2, pH 7.4) containing 0.01% (v/v) Tween-20 at room temperature for 1 h and incubated at 4 °C for 16 h with rabbit monoclonal anti-LC3B (Sigma; 1:4000), mouse anti-β-actin (Sigma; 1:5000), rabbit anti-Cathepsin D (Abcam; 1:5000), rabbit anti-Lamp-1 (Abcam; 1:1000) or rabbit anti-GAPDH (Abcam; 1:5000). Each blot was washed three times and incubated at room temperature for 1 h with the appropriate horseradish peroxidase-conjugated secondary antibody (Santa Cruz Biotechnology; 1:5000). The blots were visualized with ECL western blotting detection reagents (Millipore; WBKLS0500) and visualized with a Luminescent Image Analyzer (LAS4000, Fujifilm).
6
Autophagy Regulation via V-ATPase Complexes
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Western blot was utilized to analyze the expression of LC3 and p62/SQSTMI. Rapamycin (100 nm , abcam) and chloroquine (10 µm , Cell Signaling Technology) were used to induce and inhibit autophagy, respectively. The primary antibodies included rabbit anti‐LC3, rabbit anti‐p62/SQSTMI, rabbit anti‐LAMP1 (Abcam) and mouse anti‐β‐actin antibodies (Proteintech), secondary antibodies were goat anti‐rabbit and goat anti‐mouse antibodies (Beyotime). Gray values of protein bands were quantified by ImageJ software.
Related V‐ATPase complexes (V0 and V1 sectors) were also detected by Western blot. IEC‐6 cells were pre‐incubated with bafilomycin A1 (a specific inhibitor of V0‐sector, 100 nm ) for 1 h. The lysosome‐related organelles were separated by a Lysosome Isolation Kit (Sigma‐Aldrich) used for Western blot. The primary antibodies included rabbit anti‐ATP6V0D, anti‐ATP6V1D (Abcam), anti‐Phospho‐mTOR (Ser2448) (Cell Signaling Technology), and mouse anti‐β‐actin antibodies (Proteintech), secondary antibodies were goat anti‐rabbit and goat anti‐mouse antibodies (Beyotime). Gray values of protein bands were quantified by ImageJ software.
Related V‐ATPase complexes (V0 and V1 sectors) were also detected by Western blot. IEC‐6 cells were pre‐incubated with bafilomycin A1 (a specific inhibitor of V0‐sector, 100 n
7
Antibody Characterization of Neuroinflammation
8
Immunofluorescence Analysis of Autophagy Markers
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Primary antibodies were as follows: mouse anti-a2V (Covance, Denver, PA); rabbit anti-GAPDH (Cell signaling, Danvers, MA); rat anti-F4/80, rabbit anti-LC3B, rabbit anti-LAMP-1, rat anti-LAMP-2, rabbit anti-ATG3, rabbit anti-ATG4, rabbit anti-ATG7, rabbit anti-NF-κB p65 (all from Abcam). Secondary antibodies were as follows: goat anti-rabbit IgG-FITC, -mouse IgG AF-594, -rabbit IgG AF-594 (Invitrogen), rabbit anti-rat IgG-FITC (Abcam), goat anti-rabbit, -rat, -mouse IgG-HRP (Santa Cruz Biotechnology), donkey anti-rabbit IRDye-800CW (LI-COR Bioscience, Lincoln, NE) and EnVision + dual link System-Horseradish peroxidase (HRP) (Dako). Isotype control antibodies were as follows: control mouse IgG (R&D Systems); rat IgG isotype, mouse IgG isotype and rabbit IgG isotype (Abcam).
9
Immunofluorescence Microscopy of Transduced Cells
10
Immunofluorescent Labeling of Cellular Organelles and Tau Pathology
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For immunofluorescent labeling, cells were rinsed once with warm PBS and immediately fixed in 4% PFA in PBS for 10 min at RT. After washing twice with Tris‐buffered saline (TBS), cells were permeabilized using 0.1% Triton X‐100 in TBS for 20 min at RT, blocked in 3–5% normal goat serum (NGS) in PBS for 1 h at room temperature, and then incubated with primary antibodies (rabbit anti‐Lamp1, Abcam; rabbit anti Rab5, SantaCruz; rabbit anti KDEL, Abcam; dilution 1:250 in 3% NGS/PBS) overnight at 4°C. After washing three times with TBS, secondary antibodies were applied for 1.5 h at room temperature (diluted 1:1,000 in 3% NGS/PBS), then washed off three times with PBS, and mounted with anti‐fade mounting media containing DAPI. Human paraffin‐embedded brain slices prepared from an old individual (88 years old, female, mild neurofibrillary tangle pathology in cortex and hippocampus, Braak stage III) were first immunolabeled as described above using a mix of rabbit phospho‐tau antibodies (rabbit anti pT181, pS199, pT205, pT231, pS409; Life Technologies) and then incubated for 20 min with 0.1% (w/v) Sudan Black solution. Neurofibrillary tangles were labeled using 0.025% thioflavin‐S in 50% ethanol for 8 min followed by washing with 80% ethanol for 2 min and three times water. Imaging was performed on a Zeiss Axiovert200.
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