SM and GM1 were visualized by insertion of the fluorescent lipid analogs BODIPY-SM (0.6 µM for 20 min at RT) or -GM1 (1 µM for 20 min at 37 °C) into the PM of RBCs spread onto PLL-coated coverslips, as in [47 (link)]. Endogenous chol was decorated by the mCherry-Theta toxin fragment (0.6–1 µM for 20 min at RT) on RBCs in suspension and then spread onto PLL-coated coverslips, as in [58 (link)]. All coverslips were then placed upside down in LabTek chambers (Thermo Fisher Scientific, Waltham, MA, USA) filled with DMEM and observed with a Zeiss widefield fluorescence microscope as above. For quantification, the total number of lipid domains was assessed by manual counting on fluorescence images (purple insets in Figure S2B ) and reported to the hemi-RBC projected area determined with the Fiji software (white dotted circles in Figure S2B ). In some experiments, the abundances of chol-enriched domains in the rim and in the dimple were determined separately (white arrows and arrowheads in Figure S2B ).
Wide field fluorescence microscope
Manufactured by Zeiss
Sourced in Germany
The Wide-field fluorescence microscope is a laboratory instrument designed to visualize fluorescently labeled samples. It utilizes a wide-field illumination technique to simultaneously excite and capture the fluorescence emission from the entire sample, enabling rapid imaging of large areas.
Automatically generated - may contain errors
Lab products found in correlation
Lab tek chamber, by Thermo Fisher Scientific (1 mentions)
Axio imager widefield fluorescence microscope, by Zeiss (1 mentions)
Imagej, by Olympus (1 mentions)
Wheat germ agglutinin (wga), by Merck Group (1 mentions)
Paraformaldehyde (pfa), by Wuhan Servicebio Technology (1 mentions)
Goat serum, by Thermo Fisher Scientific (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Wuhan Servicebio Technology (1 mentions)
Xylene, by Thermo Fisher Scientific (1 mentions)
Prolong gold antifade reagent with dapi, by Thermo Fisher Scientific (1 mentions)
Diva decloaker, by Biocare Medical (1 mentions)
Confocal microscope, by Zeiss (1 mentions)
Vt1000, by Leica camera (1 mentions)
Anti egfp, by Abcam (1 mentions)
Anti camkii, by Santa Cruz Biotechnology (1 mentions)
Alexa fluor conjugated secondary antibody, by Thermo Fisher Scientific (1 mentions)
Lsm 710 confocal microscope, by Zeiss (1 mentions)
Vectashield mounting medium with dapi, by Vector Laboratories (1 mentions)
Rabbit anti cx43, by Merck Group (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Beyotime (1 mentions)
Anti fade reagent, by Agilent Technologies (1 mentions)
17 protocols using wide field fluorescence microscope
1
Fluorescent Lipid Visualization in RBCs
2
Quantifying Cell Morphology via Microscopy
3
Investigating Retromer Assembly and Cargo Glycosylation
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Myc-tagged human SFT2D2, ZDHHC5, and GRINA constructs were purchased from Origene and used for transient transfections in HeLa cells including into cells stably expressing GFP- or mStrawberry-tagged Rab or EHD1 constructs. Immunolabeled cells were imaged on a wide-field fluorescence microscope (Zeiss). siRNA-mediated silencing was performed using ON TARGETplus siRNA pools. Retromer assembly and interactions were investigated by immunoprecipitation using cells stably expressing VPS29-GFP and published protocols (Harbour et al., 2012 (link)). Cargo protein glycosylation changes upon protein silencing were assessed using agarose-bound wheat germ agglutinin (Sigma) precipitation followed by western blotting.
Supplemental Experimental Procedures , including screening details, hit selection criteria, and antibodies used are in Supplemental Information . Screening data not available in Tables S1 , S2 , S3 , and S4 , as well as images from any of the reported screens, are available upon request (syab2@cam.ac.uk).
4
Immunostaining of Cells with FITC-PPL-C
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Cells were cultured on microscope slides and washed three times in PBS for 15 minutes each. Then fixed the cells with 4% paraformaldehyde at room temperature for 15 minutes. After that, cells were treated with FITC-conjugated PPL-C or a control solution at room temperature for 2 hours. Following the treatment, the cells underwent three additional washes in PBS, each lasting 30 minutes. Images of the cells were captured using a wide-field fluorescence microscope (Zeiss in Jena, Germany), following an additional cleaning procedure.
5
Quantification of Lysosomal Markers in Cells
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The cells were seeded on polylysine-coated coverslips and treated with the indicated reagents (IFN-γ, Baf A1, and rapamycin) for various time intervals. Subsequently, the cells were fixed with 4% paraformaldehyde (Servicebio, Wuhan, China) for 15 min at room temperature and then permeabilized with 0.3% Triton X-100 (Standard Reagent, Hyderabad, India) in phosphate-buffered saline (PBS) for 10 min. After fixation and permeabilization, the cells were blocked with 5% goat serum (Invitrogen, Carlsbad, CA, USA) in PBS for 30 min at room temperature. The cells were then incubated with primary antibodies at 4°C overnight, followed by incubation with fluorescent conjugated secondary antibodies. Finally, all cells were stained with DAPI (Servicebio, Wuhan, China). The cells were visualized using a wide-field fluorescence microscope (Carl Zeiss, Baden-Württemberg, Germany). The number of LAMP-1-positive puncta was quantified using the Analyze Particle plugin for FIJI (NIH, MD, USA) after creating binary images. We analyzed three randomly selected images from each culture condition.
6
Immunofluorescent Staining of FFPE Tissues
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Formalin-fixed paraffin-embedded tissues were prepared at the TNPRC. Deparaffinization was achieved by incubation 3x in xylene (Fisher Scientific, Pittsburgh, PA) 5 min, 3x in 100% EtOH for 2 min, once in 95% EtOH (Sigma-Aldrich St. Louis, MO) 2 min, once in 80% EtOH for 2 min, 2x in distilled water for 2 min. For antigen retrieval slides were incubated 20 min in 1x Diva Decloaker (Biocare Medical, Concord, CA) at 98°C. Tissues were incubated 40 min in blocking buffer (PBS, 0.2% Fish Skin Gelatin, 10% normal goat serum, 1% BSA), washed in washing buffer (PBS, 0.2% Fish Skin Gelatin, 0.1% Triton X-100) and incubated 1 h at RT in the dark with the antibodies diluted in blocking buffer: 1∶100 AF488 clone MECA-79 and 1∶100 AF568 anti-MAdCAM-1 (clone 314G8; AbDSerotech, Raleigh, NC) or 1∶100 AF568 conjugated Mouse IgG1 Negative Control (AbD Serotec, Raleigh, NC) and Rat IgM Isotype Control (eBioscience, San Diego, CA). Slides were washed 3x with and 4x with PBS, 0.2% Fish Skin Gelatin. ProLong Gold Antifade Reagent with DAPI (Molecular Probes, Grand Island, NY) was used to detect nuclei. Slides were viewed using a wide-field fluorescence microscope (Zeiss, Thornwood, NY) and processed using ImageJ software (NIH, Bethesda, MD).
7
Quantifying Neuronal and Macrophage Density
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Immunostained tissues were imaged using a Zeiss widefield fluorescence microscope or Zeiss confocal microscope. For quantification of neuron or macrophage density, a minimum of 3 random images were taken from each tissue sample. Images were analyzed using FIJI ImageJ. Neuronal and macrophage cell bodies, per high-power field, were counted manually. The area covered by neuronal staining was measured by subtracting the background, then thresholding images, and recording the percentage of pixels covered by HuC/D staining. The average of the three images was used as a single data point per mouse. The length of macrophage projections was measured using the line tool on FIJI ImageJ. Quantification was measured per animal. The macrophage area was quantified in flattened confocal stacks via thresholding and region of interest identification.
8
Immunohistochemical Characterization of Brain Slices
9
Immunostaining of Cardiac Gap Junctions
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The atrial tissues were embedded in opti-mum cutting temperature compound (OCT compound). The embedded specimens were then cut into 8-um sections and fixed in 4% paraformaldehyde buffer, followed by immunostaining. After blocked with 5% goat serum, the sections were incubated overnight at 4ºC with the following primary antibodies: rabbit anti-Cx40 (1:50, Millipore, MA, USA), rabbit anti-Cx43 (1:100, Millipore, MA, USA) and mouse anti-αactinin antibody (1:50, BosterBio, Wuhan, China). On the next day, the samples were washed, and then incubated for 4 hours at room temperature with the following corresponding secondary antibodies: goat anti-rabbit IgG conjugated to FITC (1:100, Proteintech, Chicago, USA) and goat anti-mouse IgG conjugated to TRITC (1:100, BosterBio, Wuhan, China). Nuclei were counter stained using 4’6-diamidino-2-phenylindole (DAPI, Beyotime, Shanghai, China). Photographic images were obtained using a wide-field fluorescence microscope (Carl Zeiss microscopy, Jena, Germany).
10
Immunofluorescence Analysis of GR and FLI1
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A673 cells were serum starved for 16 hours followed by a 60 min stimulus with dexamethasone (1μM) or RU486 (1μM), as indicated. Cells were then fixed with PFA (4%) for 15 minutes, washed with PBS, permeabilized with PBS/Triton X-100 (0.01%) for 15 minutes and hybridized with primary antibodies against FLI1 (1:50) and GR (1:50; overnight). Next, the cells were incubated with secondary antibodies against Rabbit PLUS (DUO92002) and against Mouse MINUS (DUO92002) and processed using the Duolink In Situ Detection Kit (red) containing a tetramethylrhodamine-5-isothiocyanate probe (Sigma-Aldrich). Thereafter, cells were hybridized with phalloidin-FITC and DAPI for counterstaining. Coverslips were washed and placed, cells face down, onto drops of an anti-fade reagent (from Dako). Samples were examined using a widefield fluorescence microscope (Zeiss). Red dots and nuclei were counted and the number of positive stains per cells was calculated from at least 5 non-overlapping microscope fields. One-way ANOVA with Tukey correction was performed.
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