Anti‐p130cas, anti‐myosin light chain kinase (MLC), anti‐phospho p130Cas (Y165), and anti‐phosphopaxillin (Y118) antibodies were from Cell Signaling Tech., Danvers, MA, USA. Anti‐paxillin antibody was from ECM Biosciences LLC, Versailles, KY, USA, Anti‐FAK (focal adhesion kinase), anti‐vinculin, anti‐phosphotyrosine, and anti‐phospho MLC (Thr18/Ser19 serine) antibodies were from Santa Cruz Biotechnology, Dallas, TX, USA. Anti‐phospho‐vinculin (Y1065) was from abcam, Cambridge, MA, USA. Alexa Fluor 546‐conjugated secondary antibody, Alexa Fluor 488‐conjugated phalloıdin, Fluorescein isothiocyante (FITC)‐labeled dextran and ROCK inhibitor y27632 were purchased from Thermo Fisher Scientific, Waltham, MA, USA. RhoA inhibitor I, (purified C3 Transferase covalently linked to a proprietary cell penetrating moiety), was purchased from Cytoskeleton Inc., Denver, CO, USA. HMEC‐1 (human microvascular endothelial cell‐1) 10 was obtained from the Centers for Disease Control and Prevention (Atlanta, GA) and cultured in vascular cell basal medium supplemented with endothelial cell growth factor kit (Invitrogen, Waltham, MA, USA). Dasatinib was obtained from Selleck Chem., Houston, TX, USA.
Alexa fluor 546 conjugated secondary antibody
Manufactured by Thermo Fisher Scientific
Sourced in United States
Alexa Fluor 546-conjugated secondary antibody is a fluorescent-labeled detection reagent used in immunoassays and microscopy applications. It binds to the Fc region of primary antibodies, allowing for the visualization and detection of target molecules.
Automatically generated - may contain errors
Lab products found in correlation
Alexa fluor 488, by Thermo Fisher Scientific (6 mentions)
Lsm 780, by Zeiss (4 mentions)
Lsm 700, by Zeiss (4 mentions)
Lsm 710 confocal microscope, by Zeiss (4 mentions)
4 6 diamidino 2 phenylindole (dapi), by Vector Laboratories (3 mentions)
Alexa fluor 488 and, by Thermo Fisher Scientific (3 mentions)
Trypsin edta, by Thermo Fisher Scientific (3 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (2 mentions)
Prolong gold antifade reagent with dapi, by Thermo Fisher Scientific (2 mentions)
Lsm 510, by Zeiss (2 mentions)
Lsm880 airyscan elyra microscope, by Zeiss (2 mentions)
Hoechst 33258 solution, by Merck Group (2 mentions)
Bovine serum albumin (bsa), by Thermo Fisher Scientific (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (2 mentions)
Lipofectamine 3000, by Thermo Fisher Scientific (2 mentions)
Dasatinib, by Selleck Chemicals (1 mentions)
Anti vinculin, by Santa Cruz Biotechnology (1 mentions)
Anti fak, by Santa Cruz Biotechnology (1 mentions)
Anti p130cas, by Cell Signaling Technology (1 mentions)
Anti phosphotyrosine, by Santa Cruz Biotechnology (1 mentions)
49 protocols using alexa fluor 546 conjugated secondary antibody
1
Investigating Focal Adhesion Dynamics in Endothelial Cells
2
Immunofluorescent Visualization of Blastocystis
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Four weeks PI, ST4- or ST3-infected rats were euthanized and the intestine was prepared as “Swiss rolls” of 4 cm and fixed by incubating in PFA 4% during 24 h. Then, the rolls were transferred in sucrose 30% during 24–48 h at 4°C. Finally, the rolls were included in OCT compound (CellPath, Newton, United Kingdom). Sections of OCT-embedded intestine were incubated with 5% milk in PBS for 1 h, and then washed in 0.1% Triton-X100 in PBS. Anti-Blastocystis mouse polyclonal antibodies (diluted 1:200) were added for 1 h at RT, washed, and incubated further 1 h at RT with AlexaFluor 546-conjugated secondary antibody (diluted 1:1000) (anti-mouse IgG, Thermo Fisher Scientific, Waltham, MS, USA). Sections were washed and then incubated with fluorescein phalloidin (Thermo Fisher Scientific, Waltham, MS, USA) diluted 1:100 in PBS during 20 min. Finally, sections were washed and stained with DAPI. For each ST, six sections of duodenum, jejunum, ileum, caecum and colon from three rats were observed.
3
Retinal Ganglion Cell Visualization
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Enucleated eyes were immersed in 4% PFA for 2 h at 4 °C, followed by permeabilization with 0.3% Triton-X100 for 3 h at room temperature and blocking in 1% bovine serum albumin (BSA) for 2 h at room temperature. Then, retinas were incubated with RBPMS antibody (GTX118619, GeneTex) overnight at 4 °C. The next day, retinas were returned to room temperature and incubated with Alexa Fluor 546-conjugated secondary antibody (A10036; Thermo) for 1 h at room temperature. Nuclei were stained with DAPI (Vector Laboratories, Peterborough, United Kingdom). Images were captured with a Leica confocal imaging system, and ImageJ was used to count RGCs.
4
Immunofluorescence Staining of Testicular and Sperm Samples
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The testicular slides were dewaxed and then incubated in 10 mM sodium citrate buffer (pH 6.0) for 20 min in a 120 °C water bath to retrieve antigens. The slides were rinsed with TBS containing 0.1% Tween-20 and were blocked with goat serum for 60 min. Subsequently, the slides were incubated with anti-DKK3/REIC primary antibody (made by Okayama University) [11 (link)] overnight at 4 °C. The next day, the slides were rinsed with PBST and incubated with Alexa Fluor 546-conjugated secondary antibody (Thermo Fisher Scientific, Waltham, MA, USA) at room temperature for 40 min. After washing, the slides were treated with DAPI (Dojindo, Kumamoto, Japan) and then observed under LSM 780 confocal laser scanning microscope (Carl Zeiss Microscopy, Jena, Germany).
For the immunofluorescence staining of sperm, the epididymal sperm were extracted and made into sperm smears, which were subsequently formalin-fixed for 40 min at room temperature. Antigen blocking was performed using goat serum for 60 min. The smears were then incubated in anti-DKK3/REIC primary antibody overnight at 4 °C. Normal goat IgG was applied as a negative control (Figure S2 ). The use of secondary antibody and observation procedures were the same as with the testicular slides.
For the immunofluorescence staining of sperm, the epididymal sperm were extracted and made into sperm smears, which were subsequently formalin-fixed for 40 min at room temperature. Antigen blocking was performed using goat serum for 60 min. The smears were then incubated in anti-DKK3/REIC primary antibody overnight at 4 °C. Normal goat IgG was applied as a negative control (
5
Immunofluorescence Analysis of Sperm Proteins
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Spermatozoa were collected from the cauda epididymis, diluted in PBS, spotted onto slides, and air dried. The samples were then fixed with 4% paraformaldehyde for 10 min. After washing with PBS for 5 min, the slides were blocked with 5% bovine serum albumin and 10% goat serum in PBS for 1 h at room temperature. The slides were then incubated with anti-PA antibody (1:100, #012–25863, FUJIFILM Wako Pure Chemical) or anti-PPP3R2 antibody (1:100, #14005–1-AP, Proteintech) overnight at 4 °C and washed with PBS three times for 10 min each. After incubation with Alexa Fluor 546–conjugated secondary antibody (1:200, #A11081 or #A11071, Thermo Fisher Scientific) at room temperature for 2 h, the slides were washed with PBS three times for 10 min each. The slides were observed with an Olympus BX-53 microscope.
6
Immunofluorescent Analysis of Type II Collagen
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To confirm type II collagen deposition in the chondrogenically induced micromasses, 5 μm paraffin sections were obtained as described above and incubated in citrate buffer (pH 6.0) at 96°C for 40 minutes. After blocking with 10% BSA for 1 h, the sections were incubated overnight at 4°C with an anti-collagen type II primary antibody (sc-288887, Santa Cruz Biotechnology) diluted 1 : 50 in Tris-buffered saline (TBS) with 1% BSA. Subsequently, the slides were washed three times with TBS and incubated for 2 h with an Alexa Fluor 546-conjugated secondary antibody (Life Technologies, Thermo Fisher) in the dark at room temperature. The nuclei were stained with 1 μg/mL DAPI solution (sc-3598, Santa Cruz Biotechnology). Fluorescence images were obtained using a Leica TCS SP5 laser scanning confocal microscope (Leica Microsystems, Wetzlar, GER).
7
OPN4 Lentiviral Transduction and Immunofluorescence
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The HEK-293T cells or PAN T cells were infected with pCDH-Flag-OPN4 lentivirus. After 48 hours of infection, the cells were fixed in 4% paraformaldehyde, then blocked and incubated with anti-Flag antibody (Origene, diluted 1:1000, for OPN4 staining) followed by Alexa Fluor 546-conjugated secondary antibody (Thermo Fisher, diluted 1:200) incubation. Cells were further incubated with 4',6-diamidino-2-phenylindole (Sigma, 50 μg/ml) to stain the nuclei. Finally, the stained cells were visualized by confocal microscope (Zeiss LSM780).
8
Quantifying Cellular Proliferation in 3D Acini
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For immunofluorescence, cells were fixed with 4% PFA at room temperature, permeabilized with pre-chilled 0.5% Triton X-100 for 10 min prior to blocking with 2% BSA/PBS for 1 hour. After blocking, cells were incubated overnight at 4°C with primary Ki-67 antibody (Cell Signaling Technology, 9129) in a humidified chamber. The following day cells were incubated with fluorescently labeled secondary antibodies Alexa Fluor 546-conjugated secondary antibody (Thermo Fischer Scientific, A-11003) and Phalloidin 633 (Abcam, ab176758) to visualize Ki-67 and F-actin, respectively, 1% BSA/PBS for 1 hour. Slides were mounted using Prolong Gold anti-fade reagent with DAPI (Invitrogen, D1306). Images were captured using a Zeiss AxioObserver microscope equipped with a Yokogawa CSU-W1 spinning disk unit (Intelligent Imaging Innovations) and a 40x oil objective. Serial z stacks of the acini structures were acquired at 5 um intervals (usually 10-15 sections per field), and then analyzed with a custom MATLAB script (2017b, The Mathworks Inc.). Images were resampled to isotropic resolution and each spheroid was manually segmented. The DAPI signal was thresholded using Otsu’s method (Otsu, 1979 ) following intensity depth correction and smoothing. Holes were then filled and small, non-cellular objects were removed. The resulting binary nuclei image was used as a mask to measure cellular proliferation.
9
Quantifying HER2 Expression in Breast Cancer Cells
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Cells (0.5 × 105) were seeded on glass coverslips in a 24-well plate and incubated with 0.02 mg mL−1 of MNP-HC or MNP-IgG or corresponding concentrations of TZ for the indicated time points at 37 °C. When required, treatment was stopped after 48 h and cells were cultured for additional 96 h in fresh medium. At the indicated time points, cells were washed three times with PBS, fixed for 5 min with 4% paraformaldehyde (Sigma-Aldrich), permeabilized for 10 min with 0.1% Triton X-100, and incubated in 2% BSA, 2% goat serum in PBS for 2 h at room temperature. For analysis of cell uptake, MNP-HC were detected by Alexa Fluor 488-conjugated anti-human secondary antibody for 2 h at room temperature. For analysis of HER2 membrane expression, cells were immunodecorated overnight at 4 °C with anti-HER2/ErbB2 antibody (clone 29D8, Cell Signaling Technology, Inc.) diluted in blocking buffer, washed three times with PBS and incubated with Alexa Fluor 546-conjugated secondary antibody (Thermo Fisher Scientific) for 2 h at room temperature. Nuclei were stained with DAPI (0.1 μg mL−1). Coverslips were mounted in Prolong Gold antifade reagent (Thermo Fisher Scientific) and images were acquired with Leica SP8 microscope confocal system equipped with laser excitation lines 405 nm, 488 nm, 535 nm and 633 nm. Images were acquired with 63 × magnification oil immersion lens.
10
Immunofluorescence Staining of Sca-1+ Cells
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Immunofluorescence staining was done as reported [30 (link)]. Briefly, the Sca-1+ enriched cells were fixed with 4% paraformaldehyde for 15 min at room temperature and stained with anti-NR4A2 or anti-P65 antibodies, then Alexa Fluor 546-conjugated secondary antibody (Molecular Probes, Eugene, OR, USA). DAPI was used to stain the nucleus of cells. Laser scanning confocal microscopy (Leica, Wetzlar, Germany) was used for fluorescence detection. Images are representative of three independent experiments.
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