goat anti lamp1  (R&D Systems)

Journal: The Journal of Cell Biology

Article Title: N-cadherin dynamically regulates pediatric glioma cell migration in complex environments

doi: 10.1083/jcb.202401057

Figure Lengend Snippet: N-cad endocytosis, recycling, and surface levels in leader and follower cells. (A) Schematic diagram of the N-cad antibody (Ab) internalization assay. Cells were labeled with N-cad ECD Ab in the cold, warmed for various times to allow internalization, fixed, and surface and internalized Ab detected with different fluorescent secondaries. (B) Immunofluorescence detection of internalized N-cad Ab with EEA1, Rab5, or Rab4 in leader cells after 40 min internalization. Arrowheads indicate colocalization. Scale bars, 10 or 2 μm (inset). (C) Object-based colocalization quantification of internalized N-cad Ab with EEA1, Rab5, Rab4, Rab11, LAMP1, or GM130. N = 5–10 spheroids, three experiments. (D) Surface N-cad Ab increases between 40 and 60 min; a.u., arbitrary units. Two-way ANOVA Šídák’s multiple comparisons test multiple comparisons test. *P < 0.05, ***P < 0.001. N = 3–4 spheroids, three experiments. (E and F) Schematic and representative images of N-cad recycling assay. Cells were incubated with N-cad ECD antibody in the cold and warmed for 40 min to allow internalization. N-cad Ab remaining on the surface was blocked in the cold with excess F(ab’) 2 . Cells were then warmed to allow recycling before fixation and detection of surface and internalized Ab with different fluorescent secondaries. Scale bars, 10 μm. (G and H) Schematic diagram and representative images showing photoconversion of histone H2B-Dendra2 in leader and follower cells. (I–K) Flow cytometry. (I) Definition of leader, follower and spheroid cells according to extent of photoconversion. (J) N-cad surface intensity histograms. (K) Normalized median N-cad fluorescent intensity. Paired t test. *P = 0.0374. N = 3 experiments. Error bars show mean ± SEM.

Article Snippet: Goat anti-LAMP1 , Santa Cruz , SC8098 , IF (1:500).

Techniques: Labeling, Immunofluorescence, Incubation, Flow Cytometry

Journal: The Journal of Cell Biology

Article Title: N-cadherin dynamically regulates pediatric glioma cell migration in complex environments

doi: 10.1083/jcb.202401057

Figure Lengend Snippet: Antibodies used in this study

Article Snippet: Goat anti-LAMP1 , Santa Cruz , SC8098 , IF (1:500).

Techniques:

Journal: The Journal of Cell Biology

Article Title: N-cadherin dynamically regulates pediatric glioma cell migration in complex environments

doi: 10.1083/jcb.202401057

Figure Lengend Snippet: Antibodies used in this study

Article Snippet: Goat anti-LAMP1 , Santa Cruz , SC8098 , IF (1:500).

Techniques:

Journal: RSC Advances

Article Title: Development of a cationic polyethyleneimine-poly(lactic- co -glycolic acid) nanoparticle system for enhanced intracellular delivery of biologics †

doi: 10.1039/d3ra06050k

Figure Lengend Snippet: 10% PEI-PLGA nanoparticles exhibit enhanced cellular uptake in comparison to PLGA nanoparticles. Nanoparticle internalisation was assessed in HeLa cells by confocal microscopy. Cells were seed at 50 000 cells per well in an 8-well chamber slide and left overnight to adhere. Cells were treated with 0.2 mg mL −1 of various nanoparticles (PLGA, 10% PEI-PLGA, vNAR PLGA and vNAR 10% PEI-PLGA) and relevant controls for 1 h. Post treatment, cells were washed with both PBS and an acid strip wash before fixing cells for permeabilization and staining. Lysosomal regions were stained using anti-LAMP1 antibody (red) and nuclear regions stained (blue) using DAPI. FITC conjugated vNAR entrapped within the nanoparticle core can be visualised in green. Images representative of three independent experiments. Scale bars = 10 μm.

Article Snippet: After fixation and permeabilization, cells were blocked overnight at 4 °C in blocking buffer (10% Goat Serum v/v and 1% w/v BSA in PBS) before incubating with the relevant primary antibody: goat anti-mouse anti-lysosomal-associated membrane protein 1 (LAMP1) antibody (Abcam (ab25630); 1 : 50 dilution in blocking buffer) or goat anti-rabbit EEA1 antibody (cell signalling technology (c45b10); 1 : 50 dilution in blocking buffer).

Techniques: Comparison, Confocal Microscopy, Stripping Membranes, Staining

Journal: RSC Advances

Article Title: Development of a cationic polyethyleneimine-poly(lactic- co -glycolic acid) nanoparticle system for enhanced intracellular delivery of biologics †

doi: 10.1039/d3ra06050k

Figure Lengend Snippet: Assessment of nanoparticle localisation within the cell. Localisation of nanoparticles following treatment was assessed in HeLa cells using confocal microscopy. Cells were seeded at 50 000 cells per well in an 8-well chamber slide and left overnight to adhere. Cells were treated with 0.2 mg mL −1 of either vNAR PLGA or vNAR 10% PEI-PLGA nanoparticles for 1 h. Post treatment, cells were washed with both PBS and an acid strip wash before fixing cells for permeabilization and staining. Lysosomal and endosomal regions were stained using anti-LAMP1/anti-EEA1 antibody respectively, visualised in red. Nuclear regions were stained using DAPI, visualised in blue. FITC conjugated vNAR entrapped within the nanoparticle core can be visualised in green. Images representative of three independent experiments. Scale bars = 10 μm.

Article Snippet: After fixation and permeabilization, cells were blocked overnight at 4 °C in blocking buffer (10% Goat Serum v/v and 1% w/v BSA in PBS) before incubating with the relevant primary antibody: goat anti-mouse anti-lysosomal-associated membrane protein 1 (LAMP1) antibody (Abcam (ab25630); 1 : 50 dilution in blocking buffer) or goat anti-rabbit EEA1 antibody (cell signalling technology (c45b10); 1 : 50 dilution in blocking buffer).

Techniques: Confocal Microscopy, Stripping Membranes, Staining

Journal: Cells

Article Title: Megalin Orchestrates FcRn Endocytosis and Trafficking.

doi: 10.3390/cells12010053

Figure Lengend Snippet: Figure 3. Endocytosis induced movement of megalin and FcRn into clathrin vesicles and early endosomes bypassing lysosomes. (a) Quadruple staining of FcRn, megalin, with either clathrin for clathrin vesicles, with EEA1 for early endosomes, with Rab11 for recycling endosomes and with LAMP1 for late endosomes/lysosomes without endocytosis (starved cells) or induction of endocytosis by albumin as ligand for megalin and FcRn or by lactoglobulin, a ligand only for megalin. Nuclei are

Article Snippet: Rabbit anti-GM130 (ab52649, Abcam, Cambridge, UK), mouse antiEEA1 (610457, BD Biosciences, Heidelberg, Germany), mouse anti-clathrin (CBL188, Millipore, Darmstadt, Germany), rabbit anti-Rab11 (71-5300, Invitrogen, Darmstadt, Germany), goat anti-lamp1 (sc-8098, Santa Cruz Biotechnology, Dallas, TX, USA), guinea pig antimegalin [15], goat anti-calnexin (NBP1-3774, Novus, Bio-Techne, Wiesbaden, Germany), mouse anti-FcRn (sc-393064, Santa Cruz Biotechnology, Dallas, TX, USA) and rabbit antiFcRn (sc-66893, Santa Cruz Biotechnology, Dallas, TX, USA) were used.

Techniques: Staining