Western blotting and immunofluorescent assays were performed according to corresponding standard methods [25 (link)]. The specific antibodies used were listed as follows: PTEN (Cell Signaling Technology (CST), 1:1000 dilution), GSK3β (CST, 1:800 dilution), Akt (CST, 1:800 dilution), phospho (p) Akt (Ser473) (CST, 1:500 dilution), pGSK3β (Ser9) (CST, 1:500 dilution), GAPDH (CST, 1:5000 dilution), β-catenin (Abcam, 1:50 dilution), and Alexa Fluor 594-conjugated goat anti-rabbit secondary antibody (1:200, 2 mg/mL, Invitrogen). Fluorescence images were captured by a laser scanning confocal microscope (Zeiss, Germany).
Alexa fluor 594 conjugated goat anti rabbit secondary antibody
Manufactured by Thermo Fisher Scientific
Sourced in United States
Alexa Fluor 594-conjugated goat anti-rabbit secondary antibody is a fluorescently labeled antibody that binds to rabbit primary antibodies. It is used to detect and visualize target proteins in various immunoassay applications.
Automatically generated - may contain errors
Lab products found in correlation
Alexa fluor 488 conjugated goat anti mouse secondary antibody, by Thermo Fisher Scientific (6 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (3 mentions)
Prolong gold, by Thermo Fisher Scientific (3 mentions)
Alexa fluor 488 conjugated goat anti rabbit secondary antibody, by Thermo Fisher Scientific (3 mentions)
Sytox green, by Thermo Fisher Scientific (3 mentions)
Alexa fluor 647 conjugated goat anti mouse secondary antibody, by Thermo Fisher Scientific (2 mentions)
Confocal laser scanning microscope, by Zeiss (2 mentions)
Lsm 700 confocal microscope, by Zeiss (2 mentions)
Prolong gold antifade reagent with dapi, by Thermo Fisher Scientific (2 mentions)
Axio zoom v16, by Zeiss (2 mentions)
Alexa fluor 488 conjugated goat anti rabbit, by Thermo Fisher Scientific (2 mentions)
Fluoview fv10i confocal microscope, by Olympus (2 mentions)
Hoechst 33342, by Thermo Fisher Scientific (2 mentions)
Alexa fluor 488 conjugated goat anti mouse, by Thermo Fisher Scientific (2 mentions)
Eclipse 80i fluorescence microscope, by Nikon (2 mentions)
P gsk 3β, by Abcam (1 mentions)
Gapdh, by Abcam (1 mentions)
β catenin, by Abcam (1 mentions)
Gsk 3β, by Abcam (1 mentions)
Metamorph software, by Molecular Devices (1 mentions)
32 protocols using alexa fluor 594 conjugated goat anti rabbit secondary antibody
1
Protein Quantification and Localization
2
Immunofluorescence Microscopy of Cultured Parasites
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Parasites grown in culture were collected by centrifugation, washed and fixed in paraformaldehyde as described elsewhere37 (link). Slides were incubated with primary antibodies followed by Alexa Fluor 488-conjugated goat anti-mouse secondary antibody or Alexa Fluor 594-conjugated goat anti-rabbit secondary antibody (diluted 1:400) (Invitrogen). The nuclei were stained with DAPI (10 µg/mL) and cells were observed using a Zeiss Axio imager Z1 microscope; images were captured using Metamorph software (Molecular Devices). Images were processed using ImageJ software.
3
Immunofluorescence Staining for Circulating Tumor Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were permeabilized with 0.2% Triton X-100 and then quenched with 0.3% H2O2. Following blocking with 1% bovine serum albumin (GE Healthcare Life Sciences, Logan, UT, USA) in PBS, the cells were incubated with mouse anti-epithelial cell adhesion molecule (EpCAM) antibody (1:100; Cell Signaling Technology, Inc., Danvers, MA, USA, cat. no. 2929). EpCAM signals were amplified using a Tyramide Signal Amplification system (Alexa Fluor 488-conjugated goat anti-mouse IgG; Thermo Fisher Scientific, Inc.), which was used according to the manufacturer's protocol. Cells were then incubated with rabbit anti-CD45 antibody (1:100; Cell Signaling Technology, Inc., cat. no. 13917) and Alexa Fluor 594-conjugated goat anti-rabbit secondary antibody (1:200; Invitrogen; Thermo Fisher Scientific, Inc., cat. no. A-11012). The slides were mounted using Fluoroshield with DAPI (ImmunoBioScience Corp., Mukilteo, WA, USA). Stained cells were observed and images were captured using a Nikon Eclipse Ti fluorescent microscope equipped with a 400X objective. CTCs were defined as EpCAM-positive and CD45-negative. For precise identification of CTCs, PC9 cells (EpCAM-positive) and KG-1 cells (CD45-positive) were included as positive controls in each immunofluorescence staining.
4
Immunofluorescence Staining of HeLa Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
HeLa cells were seeded onto 20 mm diameter glass coverslips and transfected. The transfected cells were fixed with 4% paraformaldehyde (in PBS) for 15 min at room temperature, washed with PBS, and permeabilized with 0.5%Triton-X 100 in PBS for 10 min, then incubated in blocking buffer (5% BSA and 5% FBS in PBS) for 1 h. Cells were sequentially stained with mouse anti-importin-β antibody (Invitrogen, catalog number MA3-070, 1:500 dilution) or rabbit anti-FLAG antibody (Cell Signaling Technology, catalog number 14793, 1:1,000 dilution) for 2 h, and with Alexa Fluor 647 conjugated goat anti-mouse secondary antibody (Invitrogen, catalog number A21237, 1:500 dilution) or Alexa Fluor 594 conjugated goat anti-rabbit secondary antibody (Invitrogen, catalog number A11037, 1:500 dilution) for 1 h. Nuclei was stained with 0.5 μg/ml DAPI (4′,6-diamidino-2-phenylindole) (Thermo Fisher Scientific) for 20 min. Slides were covered with the mounting medium (Thermo Fisher Scientific). Images were recorded with Leica confocal microscope using a 63x oil objective.
5
Spinal Microglia Immunohistochemistry Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
A standard fluorescent immunohistochemistry protocol was applied to characterize spinal microglia phenotypes. Free-floating sections were incubated in a blocking buffer consisting of 3% goat serum, 1% bovine serum albumin, and 0.25% Triton X-100 in Tris-buffered saline for 1 h at room temperature followed by overnight incubation with rabbit anti-ionized calcium-binding adaptor molecule-1 (Iba-1) polyclonal primary antibody (1:1000; Wako Chemicals) at 4°C. Sections were then incubated with Alexa Fluor 594-conjugated goat anti-rabbit secondary antibody (1:500; Invitrogen) for 1 h at room temperature followed by 5 min incubation of 4′, 6-diamidino-2-phenylindole for nuclear counterstaining (1:100,000; Sigma). Sections were mounted onto slides, then coverslipped with Vectashield mounting medium (Vector Laboratories).
6
Immunofluorescence Assay for Protein Localization
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Stable cells were seeded above the cover slip and fixed with 3.7% formaldehyde for 15 min at RT and permiabilized with 0.5% Triton-X 100 in PBS for 15 min at RT. Then, the cells were incubated with a 3% BSA blocking buffer for 1 h at 4 °C. Following overnight incubation with the primary antibody at 4 °C. Cells were then incubated with Alexa Fluor 594-conjugated goat anti-rabbit secondary antibody (Invitrogen) or Alexa Fluor 488-conjugated goat anti-mouse secondary antibody (Invitrogen) at 4 °C for 17 min. After washing steps, the cover slip was mounted and images were visualized by a confocal microscope.
7
Immunostaining of Drosophila Larval Tissues
8
Immunofluorescence Staining of Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were seeded on chamber slides overnight and fixed with 4% (v/v) paraformaldehyde for 20 min at room temperature, followed by permeabilization with 0.5% TritonX-100 in phosphate-buffered saline for 30 min. Next, cells were blocked for non-specific binding with 5% goat serum in Tween-20 and PBS for 1 h. Then, cells were stained with specific primary antibodies overnight at 4 °C and subsequently with Alexa Fluor 488-conjugated goat anti-mouse secondary antibody or Alexa Fluor 594-conjugated goat anti-rabbit secondary antibody (Invitrogen). Cell nuclei were stained with 4`,6-diamidino-2-phenylindole (DAPI). After staining, fluorescence images were acquired using an LSM700 confocal microscope (Carl Zeiss, Thornwood, NY).
9
Vitamin D3 Conversion Impairment by Coal Dust
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Vitamin D3 can’t be converted into the active form 1,25(OH)2D3 by cultured cells. RAW264.7 cells were incubated with either 200 μg/mL coal dust, 1.2 × 10–10 mg/mL 1,25(OH)2VD3, or a mixture of 200 μg/mL coal dust and 1.2 × 10–10 mg/mL 1,25(OH)2VD3, respectively, for 24 h. Subsequently, the cells were fixed with a 4% paraformaldehyde solution for 15 min. Anti-MLPH as the primary antibody, Alexa Fluor®594 conjugated goat anti-rabbit secondary antibody (Invitrogen, CA, USA) were used. DAPI dye stained for 10 min. Images were taken using a confocal laser scanning microscope (Olympus, Tokyo, Japan).
10
Immunofluorescence Staining of Cultured Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were incubated at 37 °C overnight. Cells were fixed in 3.7% formaldehyde for 15 min at RT washed three times with PBS and incubated with 0.5% Triton X-100 for 15 min at RT. Cells were then incubated in blocking buffer (3% bovine serum albumin with PBS) for 1 h at 4 °C, followed by incubation with primary antibodies at 4 °C overnight. Cells were washed three times for 5 min each, after which Alexa Fluor 488-conjugated goat anti-mouse secondary antibody (Invitrogen, diluted 1:200 in PBS) or Alexa Fluor 594-conjugated goat anti-rabbit secondary antibody (Invitrogen, diluted 1:200 in PBS) was added for 17 min at 4 °C. After three washes with Tris-buffered saline containing Tween 20, cells were mounted and analyzed by confocal microscopy.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!