After GLIFin-mediated light inactivation of F-actin as described above, cells were washed with PBS three times, fixed, and permeabilized with PBS containing 4% HCHO and 0.1% Triton X-100. After 10 min, the solution was aspirated. The fixed cells were washed with PBS three times and blocked with 1% bovine serum albumin (BSA)/PBS. After 30 min, the blocking solution was aspirated, and the fixed cells were incubated in PBS containing 1% BSA, 0.66% MeOH, Alexa Fluor 647 phalloidin (2 U/ml) (A22287, Thermo Fisher Scientific), anti–α-tubulin antibody conjugated with fluorescein isothiocyanate (FITC) (3 μg/ml; ab64503, Abcam), and 4′,6-diamidino-2-phenylindole (DAPI) (3 μg/ml; D1306, Invitrogen) at ambient temperature for 1 hour. The PBS was aspirated and replaced with fresh PBS. Fluorescence imaging was done at 405/430 to 465 nm for DAPI, 488/510 to 550 nm for FITC (tubulin), and 633/661 to 750 nm for Alexa Fluor 647 (F-actin).
A22287
Manufactured by Thermo Fisher Scientific
Sourced in United States
The A22287 is a laboratory equipment product from Thermo Fisher Scientific. It is a precision instrument designed for specific applications in scientific research and analysis. The core function of this product is to perform tasks related to its intended purpose, but a detailed description cannot be provided while maintaining an unbiased and factual approach.
Automatically generated - may contain errors
Lab products found in correlation
Alexa fluor 647 phalloidin, by Thermo Fisher Scientific (4 mentions)
D1306, by Thermo Fisher Scientific (3 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (3 mentions)
Triton x 100, by Merck Group (3 mentions)
Hoechst 33342, by Thermo Fisher Scientific (3 mentions)
Alexa fluor 568, by Thermo Fisher Scientific (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (2 mentions)
Prolong diamond antifade reagent, by Thermo Fisher Scientific (2 mentions)
A1 confocal microscope, by Nikon (2 mentions)
Fv3000rs confocal microscope, by Olympus (2 mentions)
Mab1864, by Thermo Fisher Scientific (2 mentions)
Ab64503, by Abcam (1 mentions)
Pezgs0416, by Merck Group (1 mentions)
Ab93876, by Abcam (1 mentions)
Ab15580, by Thermo Fisher Scientific (1 mentions)
Ab9361, by Abcam (1 mentions)
Ab209484, by Abcam (1 mentions)
Ab185230, by Abcam (1 mentions)
A 21245, by Thermo Fisher Scientific (1 mentions)
Paraformaldehyde (pfa), by Merck Group (1 mentions)
33 protocols using a22287
1
Multicolor Cytoskeleton Visualization
2
Immunofluorescence Staining of Bone Cells
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The decalcified samples were dehydrated in 15% sucrose/PBS solution for 2 h, 30% sucrose/PBS for 2 h, and 30% sucrose/OCT (4,583, Sakura, Torrance, CA, United States) overnight at 4°C and then embedded in OCT. Cryosections measuring 8 μm in thickness were immunofluorescence-stained following standard protocols. The primary antibodies included Runx2 (1:100, #12556, Cell Signaling Technology, Danvers, MA, United States), Osterix (1:100, ab209484, Abcam, Cambridge, United Kingdom), β-galactosidase (β-gal; 1:200, ab9361, Abcam), osteocalcin (Ocn; 1:100, ab93876, Abcam), and Sox9 (1:100, ab185230, Abcam). Alexa Fluor 488 and Alexa Fluor 568 (1:200, Invitrogen, Waltham, MA, United States) were used as secondary antibodies. DAPI (62248, Invitrogen) was used for counterstaining. ImageJ was used to analyzed the ratio of Osterix+/tdTomato + cells to Osterix + cells.
Cell samples were plated in a 4-well chamber slide (PEZGS0416, Millipore). The slides were washed with PBS, immediately fixed with 4% paraformaldehyde for 15 min, and blocked with goat serum for 30 min at room temperature. The cells were incubated with the primary antibody (Ki67, 1:200, ab15580) at 4°C overnight, then incubated with secondary antibodies at room temperature for 1 h, stained with phalloidin for 20 min (1:50, A22287, ThermoFisher Scientific), and counterstained with DAPI.
Cell samples were plated in a 4-well chamber slide (PEZGS0416, Millipore). The slides were washed with PBS, immediately fixed with 4% paraformaldehyde for 15 min, and blocked with goat serum for 30 min at room temperature. The cells were incubated with the primary antibody (Ki67, 1:200, ab15580) at 4°C overnight, then incubated with secondary antibodies at room temperature for 1 h, stained with phalloidin for 20 min (1:50, A22287, ThermoFisher Scientific), and counterstained with DAPI.
3
Immunofluorescence Microscopy of Cell Adhesion
4
Photoreceptor Outer Segment Phagocytosis
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Purification and labeling of bovine photoreceptor outer segments (POS) was performed as described previously [26 (link)]. FITC-labeled POS were incubated with mcRPE at a ratio of 20 POS per cell and mouse IgG1 for 16 h at 37 °C and 5% CO2. Microcarriers were subsequently washed five times with PBS+/+ and fixed using 4% paraformaldehyde. For function-blocking experiments, mcRPE were pre-incubated for 1 h with 30 ug/mL αvβ5 integrin antibody (Abcam, #ab177004) followed by a 16 h co-incubation of mcRPE with αvβ5 integrin antibody and FITC-labeled POS. Samples were rinsed, fixed and co-stained with phalloidin (Thermo Fisher Scientific #A22287) to label F-actin and Hoechst to label nuclei. Imaging was performed using a Leica SP8 Resonant Scanning Confocal microscope. POS particles and nuclei were thresholded and 3 representative field over-views were counted using Analyze Particles function with size restrictions; quantification was performed using FIJI ImageJ.
5
Evaluating Progerin and LC3B in CCl4-Induced Liver Fibrosis
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Hepatic paraffin sections (3 μm) of CCl4-induced liver fibrosis rat models were prepared for the IF staining for progerin, vWF, and LC3B. The primary antibodies included anti-progerin (1:50), anti-vWF (1:200), and anti-LC3B (1:200). Subsequently, the sections were exposed to the secondary antibodies and DAPI. The sections were visualized with the fluorescence microscope.
The treated LSECs in confocal dishes were fixed with 4% paraformaldehyde and then permeabilized with 0.1% Triton X-100 and blocked with 3% BSA. Next, LSECs were incubated with primary antibodies, including anti-acetyl Lysine (1:200), anti-progerin (1:50), and anti-LC3B (1:200), followed by the secondary antibodies and DAPI. Lastly, LSECs were stained with a phallotoxin (Thermo, #A22287 and #T7471) to detect F-actin. The positive cells were observed by the confocal microscopy and quantified by Image J V1.8.0 software. The experiment was repeated three times.
The treated LSECs in confocal dishes were fixed with 4% paraformaldehyde and then permeabilized with 0.1% Triton X-100 and blocked with 3% BSA. Next, LSECs were incubated with primary antibodies, including anti-acetyl Lysine (1:200), anti-progerin (1:50), and anti-LC3B (1:200), followed by the secondary antibodies and DAPI. Lastly, LSECs were stained with a phallotoxin (Thermo, #A22287 and #T7471) to detect F-actin. The positive cells were observed by the confocal microscopy and quantified by Image J V1.8.0 software. The experiment was repeated three times.
6
Quantifying Brachyury and SOX2 Co-Expression in Chicken Embryos
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Chicken embryos from stage 4 to 20HH were harvested and fixed in 4% PFA at 4°C overnight. Embryos were immunostained in whole mount using T/Brachyury, SOX2, and Alexa Fluor 647 Phalloidin (1/500 Thermo Fisher: A22287) combinations and imaged by confocal laser microscopy (LSM 780, Zeiss). SOX2/T-double positive cells were segmented and counted using ImageJ software. To identify the double-positive cells, cells in the T and SOX2 channel were manually thresholded (around 1.3% of the histogram). We used the image calculator tool from ImageJ to generate a new image by performing the image operation T AND SOX2 and identify the double-positive cells. Cell were then counted automatically by the ‘Analyze Particle’ module on ImageJ (particle size < 500 pixel square). Embryos were grouped by stage, and data is shown in Figure 5A . n = 7 at stage 4HH; n = 11 at stage 5HH; n = 6 at 5–10 somites; n = 5 at 15–20 somites; n = 5 at 30 somites; n = 4 at 35 somites and older. Total n = 38 embryos.
7
Mouse Primary Hippocampal Neurons Isolation
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Mouse primary hippocampal neurons (mPHN) were isolated from E17 to E18 C57Bl/6J (Janvier) (IMSR Cat# JAX_000664, RRID:IMSR_JAX:000664) embryos, dissociated, and plated in 96-well plates (Greiner, μClear, 655946) previously coated with poly-L-lysine (Sigma Aldrich, P1274) at 20,000 cells per well. Neurons were kept at 37 °C and 5% CO2 in B-27 and GlutaMax supplemented Neurobasal medium (Gibco, 10888022) (76 (link), 77 (link)).
QBI-HEK 293A (RRID:CVCL_6910) cells were cultured in DMEM (Gibco, 11960-044) supplemented with 10% FBS and 1% PenStrep (Gibco, 15140163). In total, 5000 cells were plated per well in 96-well plates and kept at 37 °C and 5% CO2. Forty-eight hours after plating, cells were treated with 1 μM YM-201636 and incubated for 30 min before being fixed with 4% paraformaldehyde and stained for LAMP1 (Abcam, Cat# ab25245, RRID:AB_449893 ) and F-actin (Thermo Fisher, Cat# A22287, RRID:AB_2620155 )
QBI-HEK 293A (RRID:CVCL_6910) cells were cultured in DMEM (Gibco, 11960-044) supplemented with 10% FBS and 1% PenStrep (Gibco, 15140163). In total, 5000 cells were plated per well in 96-well plates and kept at 37 °C and 5% CO2. Forty-eight hours after plating, cells were treated with 1 μM YM-201636 and incubated for 30 min before being fixed with 4% paraformaldehyde and stained for LAMP1 (Abcam, Cat# ab25245, RRID:
8
Immunofluorescence Staining of BV-2 Cells
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BV-2 cells were fixed in 2% paraformaldehyde (VVR Life Science) for 5 min, permeabilized by incubation in 0.05% Triton X-100 for 10 min, and blocked with 2.5% normal donkey serum for 1 h at room temperature (RT). The samples were then incubated with primary antibodies TNFa (1:100; AB1793, Abcam, Cambridge, MA, Unites States), COX-2 (1:100; MA5-14568, Thermo Fisher Scientific, Waltham, MA, Unites States), β-Tubulin (1:200; MA5-16308, Thermo Fisher Scientific, Waltham, MA, Unites States), or Alexa Fluor™ 647 Phalloidin conjugate (1:100; A22287, Thermo Fisher Scientific, Waltham, MA, Unites States) for 1 h at room temperature and washed with PBS-Tween 20 (0.05%; PBS-T). After PBS-T washing, they were then visualized by goat anti-rabbit IgG (H + L), Alexa Fluor 488 (A-11034, 1:1000, Fisher Scientific, Waltham, MA, Unites States), donkey anti-mouse IgG (H + L), Cyanine3 (A10521, 1:1000; Fisher Scientific, Waltham, MA, Unites States)/ The cell nuclei were visualized by incubation with 4′,6-diamidino-2-phenylindole (DAPI); (1:5000; Sigma). The slides were mounted with a ProLong Diamond antifade reagent (Thermo Fisher Scientific, Waltham, MA, Unites States).
9
Ephrin-B1 and EphB Receptor Imaging
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Mouse embryos were subjected to immunofluorescence using 2 µg/ml ephrin-B1 (AF473; R&D Systems), 2 µg/ml EphB2 (AF467; R&D Systems), 2 µg/ml EphB3 (AF432; R&D Systems), and 10 µg/ml GFP (ab13970; Abcam) antibodies. The following secondary antibodies were used: Alexa Fluor 488–conjugated donkey anti–chicken IgG (1:1,000, 703-545-155; Jackson ImmunoResearch Laboratories, Inc.) and Cy3-conjugated donkey anti–goat IgG (1:400; 705-165-003; Jackson ImmunoResearch Laboratories, Inc.). To label F-actin, Alexa Fluor 647–conjugated phalloidin (1:40; A22287; Thermo Fisher Scientific) was used. For Western blotting, the following primary antibodies were used: rabbit anti-ROCK1 (1:500; sc-5560; Santa Cruz Biotechnology, Inc.), ROCK2 (1:500; sc-5561; Santa Cruz Biotechnology, Inc.), and HSP70 (1:1,000; 610607; BD). The following IRDye secondary antibodies were used: goat anti–mouse 680RD (1:5,000; 925–68070; LI-COR Biosciences) and goat anti–rabbit 800CW (1:5,000; 926–32211; LI-COR Biosciences).
10
Immunofluorescence Staining Protocol
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Permeabilisation was performed using PBS with 0.1 M glycine, 0.3% Triton X-100 for 30 min at RT. Primary antibodies were incubated overnight at 4°C. All primary antibodies were diluted in blocking buffer (1% BSA, 0.1% Tween in PBS) at 1:200. Cells were then washed three times with 0.1% Tween-PBS before being incubated with fluorescently conjugated Alexa Fluor secondary antibodies diluted 1:500 in 0.1% Tween-PBS for 2 hr at room temperature. Where indicated Alexa Fluor 647 Phalloidin (1:100; A22287, Thermo Fisher Scientific) and DAPI (1:1000; D3571 Thermo Fisher Scientific) were added concomitantly with the secondary antibodies. Primary and secondary antibody lists can be found in Supplementary file 4 .
Imaging took place on an inverted SP5 confocal microscope (Leica Microsystems) using a Leica HC PL FLUOTAR 0.5 NA 20.0× dry objective and a Leica HC PL APO 1.4 NA 63× oil objective, or on an inverted SP8 confocal microscope (Leica Microsystems) with a Leica HC PL APO CS2 1.4 NA 63× oil objective and a Leica Fluotar VISIR 0.95 NA 25× water objective. Within each independent experiment, laser power and detector gain were maintained constant.
Imaging took place on an inverted SP5 confocal microscope (Leica Microsystems) using a Leica HC PL FLUOTAR 0.5 NA 20.0× dry objective and a Leica HC PL APO 1.4 NA 63× oil objective, or on an inverted SP8 confocal microscope (Leica Microsystems) with a Leica HC PL APO CS2 1.4 NA 63× oil objective and a Leica Fluotar VISIR 0.95 NA 25× water objective. Within each independent experiment, laser power and detector gain were maintained constant.
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