af488 α tubulin  (Proteintech)

Journal: npj Gut and Liver

Article Title: Impact of oxidized low-density lipoprotein on rat liver sinusoidal endothelial cell morphology and function

doi: 10.1038/s44355-024-00009-5

Figure Lengend Snippet: Fig. 3 | Fluorescence microscopy of rat LSECs from male rats, comparing between cells cultured in RPMI only (control) and those treated with 80 µg/mL oxLDL24. Upper row shows the merged channels stained for the cell nucleus (DAPI - blue), α-tubulin fibers (antibody against α-tubulin coupled to Alexa Fluor 488 – green), and the endocytosed FSA coupled to Alexa Fluor 647 (magenta). Pictures are representative of all imaged samples.

Article Snippet: Antibodies for immunostaining of cells were bought from Santa Cruz (anti α-tubulin AF488 and AF647, Cat. Nr. sc-23948) and Sigma-Aldrich (Phalloidin-Atto 647 N,Cat.Nr.

Techniques: Fluorescence, Microscopy, Cell Culture, Control, Staining

Journal: Biophysical Journal

Article Title: Phalloidin-PAINT: Enhanced quantitative nanoscale imaging of F-actin

doi: 10.1016/j.bpj.2024.07.003

Figure Lengend Snippet: Chaotropic perturbation enhances F-actin labeling consistency and quantification of phalloidin-PAINT. ( a ) Schematic illustration of chaotrope (KSCN) enhanced single-molecule labeling of F-actin ( top ). Relative enhancement of phalloidin-AF647 combined dissociation and photobleaching rate in the presence of KSCN ( bottom ). Error bars were determined by the propagation of error. ( b ) A representative superresolution image reconstructed from a 30,000-frame acquisition with phalloidin-AF647 in the presence of 300 mM KSCN on a U2OS cell. ( c ) Zoomed-in view of the boxed region in ( b ). A zoomed-in view of the indicated boxed region shows the thin actin fibers ( right ). ( d ) Gaussian-fitted cross-sectional profile across the F-actin fibers indicated in ( c ). ( e ) d STORM image of a phalloidin-AF647-stained U2OS cell. ( f ) The phalloidin-PAINT image of the U2OS cell shown in ( e ) performed after the d STORM acquisition. ( g ) Merged view of the boxed region shown in ( e ) and ( f ) ( top ). The zoomed-in view of the yellow boxed region indicated ( bottom ). Yellow arrowheads point to the thin actin fibers. ( h ) Linear regression fit of the cumulative number of events per 1000 frames in the presence ( squares , blue solid line fit ) and absence ( circles , orange dash line fit ) of 300 mM KSCN. ( i ) Quantitative comparison of the relative F-actin densities in stress fibers (SF) and thin fibers (TF). Error bars represent standard deviation. ( j ) Phalloidin-PAINT imaging on a U2OS cell immunostained for microtubules and mitochondria. Shown is a zoomed-in view of the phalloidin-PAINT superresolution image ( cyan ) merged with the immunofluorescence images of microtubules (DM1A-AF488, magenta ) and mitochondria (F10-AF546, green ) captured after the phalloidin-PAINT image acquisition. The corresponding full field of view merged image is shown in Fig. S4 . ( k ) Immunostaining persisted through phalloidin-PAINT image acquisition. Single-channel images of F-actin (phalloidin-PAINT, cyan ), microtubules ( magenta ), and mitochondria ( green ) shown in ( j ). ( l ) Fluorescence intensity decreased after 17 h in the phalloidin-PAINT imaging buffer. Cross-sectional intensity profiles across the indicated lines in ( k ) at time points 0 and 17 h. Blue arrowheads point to representative peaks with fluorescence intensity reduction marked. Solid line, 0 h; dashed line, 17 h. ( m ) Cross-linking of antibody staining prevented KSCN-induced fluorescence intensity loss of DM1A-488. Immunofluorescence images of the microtubules labeled with DM1A-AF488 and cross-linked to the substrate via a postfixation step before (0 h) and after (17 h) incubating with 300 mM KSCN. ( n ) Cross-sectional intensity profiles across the indicated lines in ( m ). Solid line, 0 h; Dashed line, 17 h. ( o ) Cross-linking of antibody staining prevented KSCN-induced fluorescence intensity loss of F10-AF546. Immunofluorescence images of the mitochondria labeled with F10-AF546 and cross-linked to the substrate via a postfixation step before (0 h) and after (17 h) incubating with 300 mM KSCN. ( p ) Cross-sectional intensity profiles across the indicated lines in ( o ). Solid line, 0 h; Dashed line, 17 h. ( b , c , e , and f ) Images are visualized using the “Fire” LUT. Scale bars, 10 μ m ( b ), 5 μ m ( c , left , e , f , j , k , m , n ), 2 μ m ( c , right ), and 1 μ m ( g ).

Article Snippet: Alexa Fluor 647 (AF647) Phalloidin (A22287), Alexa Fluor 568 (AF568) Phalloidin (A12380), eBioscience LPS (00497693), Alexa Fluor 488 (AF488) conjugated mouse monoclonal primary antibody against α-tubulin (clone DM1A, 53-4502-82), AF647 conjugated goat anti-mouse Superclonal recombinant secondary antibody (A28181), Cytiva HyClone HEPES Solution (SH3023701), and Cytiva HyClone Non-Essential Amino Acids 100× Solution (SH3023801) were purchased from Thermo Fisher Scientific, Waltham, MA, USA.

Techniques: Labeling, Staining, Comparison, Standard Deviation, Imaging, Immunofluorescence, Immunostaining, Fluorescence