Immunostaining was performed on whole mounted embryos from 18 to 48 hpf as specified. After anesthesia with tricaine (MS-222, Sigma-Aldrich), embryos were fixed in 4%PFA for 2 h prior to permeabilization in PBS/Triton1% for 2 h. Primary antibodies were applied overnight at 4°C at specific concentrations: anti-Synaptotagmin2 (ZNP1, DSHB, 1/100); anti-Dystrophin (MANDRA1, DSHB, 1/50); anti-MTCO1 (ab14705, Abcam, 1/300); anti-light meromyosin portion of heavy chain myosin II (MF20, DSHB, 1/20). Subsequent labeling were applied as needed for 1 h at room temperature: Phalloidin-TRICT (P1951, Sigma-Aldrich, 100 nM) and αBungarotoxin-TRITC (T0195, Sigma-Aldrich, 100 nM).
Phalloidin trict
Manufactured by Merck Group
Sourced in United States
Phalloidin-TRICT is a fluorescent dye used to label and visualize filamentous actin (F-actin) in cells. It binds specifically to F-actin and emits a red fluorescence upon excitation, allowing for the detection and localization of actin structures within a cell sample.
Automatically generated - may contain errors
Lab products found in correlation
Ab14705, by Abcam (1 mentions)
αbungarotoxin tritc, by Merck Group (1 mentions)
Ms 222, by Merck Group (1 mentions)
Phalloidin 488, by Thermo Fisher Scientific (1 mentions)
Fluoromount gtm, by Electron Microscopy Sciences (1 mentions)
Lsm 800 confocal laser scanning microscope, by Zeiss (1 mentions)
Zen blue software, by Zeiss (1 mentions)
Goat anti mouse alexa 568, by Thermo Fisher Scientific (1 mentions)
Fluoromount g, by Southern Biotech (1 mentions)
Mouse anti engrailed, by Developmental Studies Hybridoma Bank (1 mentions)
4 protocols using phalloidin trict
1
Immunostaining in Zebrafish Embryos
2
Immunostaining and Confocal Imaging of Cells
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Cells were fixed with 3.7% PFA (or 3.7% PFA + 0.5% Tx-100 in the case of ZO-1 staining) during 15 min at RT. After washing (1X PBS), cells were incubated for 15 min in permeabilization buffer (0.5% Tx100 in 1X PBS), blocked for 1 h (10% goat serum in PBS) and incubated with the corresponding primary antibodies at 4 ºC overnight (o/n) (Table 1). Then, cells were washed (1X PBS) and incubated for 45 min at RT with the secondary antibodies (Table 1). Finally, they were stained with DAPI (1 µg/ml), Phalloidin-TRICT (Sigma Aldrich P1951; 1:100 dilution) or Phalloidin-488 (Thermo Fisher Scientific A12379, 1:100) as indicated, and mounted with Fluoromount-G TM (Electron Microscopy Sciences #17984-25). The immunostaining of 3D-endothelial differentiated cells was performed following the protocol of (Fernandez-Alonso et al., 2015). Confocal microscopy images (8bits) were obtained in a Zeiss LSM800 Confocal Laser Scanning Microscope using 25x Plan-Apo/0.8 numerical aperture or 63x Plan-Apo/1.4 numerical aperture oil objectives at RT. In some cases, a 0.5 zoom was used. Confocal Z-stack images were acquired in all cases and stacks were z-projected to maximum intensity.
Images were processed with the ZEN blue software (Carl Zeiss Microscopy GmbH).
Images were processed with the ZEN blue software (Carl Zeiss Microscopy GmbH).
3
Immunohistochemical Analysis of Drosophila Larvae
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Larvae were dissected in cold phosphate buffered saline (PBS) and fixed in 4% formaldehyde in PBS for 30 min at room temperature. Larvae were then washed three times for 20 min with PBT and blocked in 2% normal donkey serum in PBT for 30 min. We incubated the tissue overnight at 4° in a primary antibody solution containing mouse anti-Engrailed (Developmental Studies Hybridoma Bank 4D9, 1:40) diluted in 2% normal donkey serum in PBT. After washing three times for 20 min in PBT, larvae were incubated in the dark with goat anti-mouse Alexa 568 (Invitrogen, 1:200) and TRICT-Phalloidin (Sigma, 1:200) diluted in 2% normal donkey serum in PBT overnight at 4°. Larvae were rinsed with PBT, and ovaries were mounted on a poly-l -lysine-coated coverslip using Fluoromount-G (SouthernBiotech).
4
Neurite Growth Assay in PC12 Cells
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NGF-differentiated PC12 cells were detached and dispersed with Pasteur pipette and re-plated onto poly-L-lysine coated 24-well plates in a medium containing 1% horse serum and 0.5% fetal calf serum. NEP1-40, a specific antagonist of NgR1, was added at a final concentration of 1 µM at 14 hours after replating. Nogo-p4 and NGF was added at 15 h and 16 h after re-plating, respectively. The cells were fixed with 4% paraformaldehyde 24 h later. To visualize neurites, PC12 cells were permeabilized with 0.2% Triton X-100 in Phosphate-Buffered Saline (PBS) for 10 min, washed with PBS, and incubated with 200 ng/ml of TRICT-phalloidin (Sigma-Aldrich, St. Louis., MO, USA, catalog no. P1951) for F-actin staining. The images were photographed at ×400 magnification. Five to 10 fields containing a total of at least 100 cells were randomly chosen for neurite growth assay. The neurite length of PC12 cells was measured using STOPSOFTWARE Version 4.6.
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