Cells were fixed for 10 min at 4°C in 4% paraformaldehyde in PBS, permeabilized with 0.1% Triton X-100 (Sigma-Aldrich, St. Louis, MO) for 10 min at room temperature, and blocked for 1 hour in PBS with 1% horse serum (Sigma-Aldrich, St. Louis, MO). The primary antibodies used were as follows: anti-NANOG (1:400; cat. no. 4903, Cell Signaling), anti-SOX17 (1:100; cat. no. 81778, Cell Signaling), anti-FOXA2 (1:100; cat. no. 685802, BioLegend), anti-SMAD2 (1:100; cat. no. 3122, Cell Signaling), anti-SMAD3 (1:100; cat. no. 9523, Cell Signaling), anti–p-SMAD2 (1:100; cat. no. 3108, Cell Signaling), and anti–p-SMAD3 (1:100; cat. no. 9520, Cell Signaling). The treated cells were subjected to three washes with PBS and further incubation with Alexa Fluor secondary antibodies (1:500; Jackson ImmunoResearch) for 1 hour at room temperature in the dark. The cells were then washed three times with PBS, with 4′,6-diamidino-2-phenylindole (Sigma-Aldrich, St. Louis, MO) added to the first wash to stain the nuclei. Images were acquired using a Confocal Zeiss LSM880. Intensity analysis was performed using ImageJ 1.51j8 (National Institutes of Health, MD, USA). For time-lapse imaging, cells were placed on an inverted microscope (Nikon, Ti-U inverted microscope system) and recorded every 2 hours for 72 hours in an environmental chamber maintained at 37°C with 5% CO2.
Alexa fluor secondary antibody
Manufactured by Jackson ImmunoResearch
Sourced in United States
Alexa Fluor secondary antibodies are fluorescently labeled antibodies designed to detect and visualize primary antibodies in immunoassays. They are available in a range of fluorescent dye conjugates to suit various detection requirements.
Automatically generated - may contain errors
Lab products found in correlation
Fluoromount g, by Southern Biotech (2 mentions)
Dp72 microscope, by Olympus (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (2 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Anti sox17, by Cell Signaling Technology (1 mentions)
Anti foxa2, by Cell Signaling Technology (1 mentions)
Ti u inverted microscope system, by Nikon (1 mentions)
Horse serum, by Merck Group (1 mentions)
Anti smad3, by Cell Signaling Technology (1 mentions)
Anti p smad2, by Cell Signaling Technology (1 mentions)
Anti p smad3, by Cell Signaling Technology (1 mentions)
Triton x 100, by Merck Group (1 mentions)
Sp8 confocal system, by Leica (1 mentions)
Anti gfp antibody, by Thermo Fisher Scientific (1 mentions)
Vectashield mounting medium, by Vector Laboratories (1 mentions)
Fv1000 microscope, by Olympus (1 mentions)
Ab195377, by Abcam (1 mentions)
Ab18180, by Abcam (1 mentions)
Normal goat serum (ngs), by Vector Laboratories (1 mentions)
Ab15580, by Abcam (1 mentions)
21 protocols using alexa fluor secondary antibody
1
Immunofluorescence Imaging and Time-Lapse Analysis of Stem Cell Markers
2
Immunofluorescence Staining and Confocal Imaging
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were fixed for 30 min with 4% paraformaldehyde (PFA), washed with 1× phosphate-buffered saline (PBS), and permeabilized with 0.5% Triton X-100/1× PBS for 30 min, blocked with 10% donkey serum for 1 h, and incubated with mouse anti-FLAG monoclonal or anti-2A primary antibodies in 0.1% Triton X-100/1× PBS overnight at 4°C. The next day, cells were washed with 1× PBS and incubated with the corresponding Alexa Fluor secondary antibodies (Jackson ImmunoResearch Laboratories; donkey anti-rabbit, anti-mouse, IgG (H+L) 488, 594, or 680) at a 1:400 dilution for 1 h at room temperature, using Hoechst 33258 (1:5,000) as a nuclear counterstain. Fluorescent confocal images were acquired and analyzed using the Leica SP8 confocal system.
3
Whole-Mount Immunostaining of Adult Brains
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Immunostaining of whole-mount adult brains was performed as described previously [97 (link)]. Dissected brains were fixed with 3.7% formaldehyde in PBS, blocked with PBS containing 0.3% Triton X-100 and 5% normal goat serum, and incubated with anti-GFP antibody (Invitrogen, A-11122; diluted at 1:1000) or anti-mCherry antibody (Developmental Studies Hybridoma Bank, DSHB-mCherry-3A11; diluted at 1:20) at 4°C overnight. After washing in PBS containing Triton X-100, immunostained samples were further incubated with species-specific Alexa Fluor secondary antibodies (Jackson ImmunoResearch) at 4°C overnight, washed with PBS containing Triton X-100, and then mounted using VECTASHIELD mounting medium (Vector Laboratories). Confocal images were obtained using an FV1000 microscope (Olympus) and analyzed using ImageJ software.
4
Immunofluorescence Staining of αSMA and Vimentin
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were cultured in confocal dishes, fixed with 4% paraformaldehyde for 15 min and permeabilized with 0.2% TritonX-100 for 10 min. Blocking was performed in 5% BSA for 30 min at room temperature. Subsequently, cells were incubated with primary antibodies overnight at 4 °C. Cells were then washed with PBS and incubated with Alexa Fluor secondary antibodies at room temperature for 1 hour. After washed with PBS, cells were mounted with DAPI staining solution. Primary antibodies targeting αSMA and vimentin were purchased from Abcam (Cambridge, UK). Besides, Alexa Fluor secondary antibodies were purchased from Jackson ImmunoResearch (West Grove, PA, USA). Finally, the fluorescence was observed by confocal laser-scanning microscope system DeltaVision (GE Healthcare, UK), and images were processed with relevant software.
5
Immunohistochemical and Immunocytofluorescence Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For immunohistochemistry (IHC) analysis, tissue slides were deparaffinized and rehydrated through an alcohol series followed by antigen retrieval with sodium citrate buffer, blocked with 5% normal goat serum (Vector) with 0.1% Triton X-100 and 3% H2O2 in PBS for 60 min at room temperature and then incubated with appropriate primary antibodies against ALKBH5(1:1000, ab195377, Abcam), ABCA1(1:200, ab18180, Abcam), and Ki67 (1:1000, ab15580, Abcam) at 4 °C overnight. IHC staining was performed with horseradish peroxidase (HRP) conjugates using DAB detection. Nuclei were counterstained with Hoechst.
For immunocytofluorescence (IF), SKCM cells were first treated with 4% PFA for 15 min. The sections and cells were blocked with Immunol Staining Blocking Buffer (Beyotime) for 1 h and incubated with the primary antibody against ABCA1(1:200, ab18180, Abcam) overnight at 4 °C, then were incubated with Alexa Fluor secondary antibodies (Jackson ImmunoResearch, PA, USA) for 1 h. This was followed by incubation with a diamidinyl phenyl indole (DAPI) coloration for 10 min (Servicebio, China). Images were taken with the fluorescence microscope (IX83, Olympus, Japan).
For immunocytofluorescence (IF), SKCM cells were first treated with 4% PFA for 15 min. The sections and cells were blocked with Immunol Staining Blocking Buffer (Beyotime) for 1 h and incubated with the primary antibody against ABCA1(1:200, ab18180, Abcam) overnight at 4 °C, then were incubated with Alexa Fluor secondary antibodies (Jackson ImmunoResearch, PA, USA) for 1 h. This was followed by incubation with a diamidinyl phenyl indole (DAPI) coloration for 10 min (Servicebio, China). Images were taken with the fluorescence microscope (IX83, Olympus, Japan).
6
Immunohistochemistry for Spinal Cord Analysis
7
Immunohistochemical Staining of Myelination
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
To prepare for antibody staining, slides were thawed then rehydrated in PBS. In order to effectively stain myelin proteins, tissue was put through ascending, then descending ethanol dilutions (50, 70, 90, 95, 100, 95, 90, 70, 50%), followed by three washes of PBS. Tissue was then blocked with 10% normal donkey serum dissolved in PBS with 0.1% Triton X-100 for 30 min. Primary antibodies were diluted in PBS with 0.1% Triton X-100 and applied to the slides overnight at room temperature in a humid chamber. The following morning, slides were washed and incubated with donkey Dylight or Alexa Fluor secondary antibodies (Jackson ImmunoResearch Laboratories, Inc.) for 2 h, then washed again before being coversliped using Fluoromount-G (Southern Biotech, 0100-01). Antibodies used were raised against the following antigens: CC1 (1:300, Millipore, OP80), OLIG2 (1:500, Millipore, AB9610), MYRF (1:300, N-terminus, generously provided by Dr. Michael Wegner), GFP (1:4000, Abcam, ab13970), GFAP (1:1000, Sigma, G3893), PDGFRα (1:200, R and D Systems, AF-307-NA), NF200 (1:1000, Sigma, N0142), SMI312 (1:1000, Covance, SMI-312R-100) and P0 (1:100, Aveslabs, PZO).
8
Immunoblotting and Immunofluorescence Antibodies
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Following primary antibodies were used for this study: rabbit anti‐nucleolin (Abcam, ab50279, 1:1,000 for WB), rabbit anti‐nucleolin (ProteinTech, 10556‐1‐AP, 1:100 for IF), rabbit anti‐Kif5a (Abcam, ab5628, 1:1,000 for WB, 1:100 for Wes), rabbit anti‐HA (Sigma, H6908, 1:1,000 for WB), rabbit anti‐LAMP1 (Abcam, ab24170, 1:1,000 for WB), mouse anti‐GAPDH (Millipore, MAB374, 1:5,000 for WB), mouse anti‐β‐III tubulin (R&D systems, MAB1195, 1:1,000 for WB), mouse β‐III tubulin (Tuj1) (BioLegend 801202, 1:500 for IF), mouse anti‐NFH (Developmental Studies Hybridoma Bank, RT97, 1:200 for IF), chicken anti‐NFH (Abcam, ab72996, 1:1,000 for IF), mouse anti‐neurofilament (BioLegend 837904, 1:1,000 for IF), and sheep anti‐digoxigenin (Roche, 11207733910, 1:2,000 for ELISA). HRP‐conjugated secondary antibodies for immunoblots were purchased from Bio‐Rad Laboratories, Alexa Fluor Secondary antibodies were purchased from Jackson ImmunoResearch, and streptavidin‐HRP was purchased from Abcam (ab7403, 1:10,000).
9
Histological and Immunohistochemical Analysis of Oocytes
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Histological and IHC protocols used in this study were adopted from our previous study39 (link). 5 μm thick deparaffinised tissue sections were incubated with primary antibodies (Table S2 ), followed by AlexaFluor secondary antibodies (1:250; Jackson ImmunoResearch Laboratories, PA, USA) for signal detection. Pictures were obtained using an Olympus DP72 microscope or Olympus FV1000 (Olympus, Tokyo, Japan) with the same gain and exposure for tissues from the control and mutant mice. For the assessment of florescence intensity of βcatenin at least 30 oocytes were counted, from three different animals from both control and mutant group, using ImageJ (National Institutes of Health, USA). β-galactosidase staining procedure is described in40 (link).
10
Western Blot Analysis of Signaling Pathways
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were harvested in 2 × Laemmli buffer (62.5 mM Tris–HCl, pH 6.8, 25% glycerol, 2% SDS, 1 mM NaF, 1 mM Na2P2O4, 1 mM NaVO4, 2.5 nM Glycerol phosphate, 1 mM PMSF). Protein concentrations were determined with BCA protein assay kit (Thermo Fisher Scientific, RE232675). 5 μg total proteins per sample were loaded on SDS-PAGE gel and transferred to PVDF membranes. The membrane was blocked in 5% non-fat-milk in 1 × TBST at room temperature for 1 h. Primary antibodies were applied overnight at 4 °C. Peroxidase-conjugated secondary antibodies (Jackson ImmunoResearch Laboratories, Alexa Fluor® secondary antibodies) were diluted 1:10,000 and applied for 1 h at room temperature. Signals were visualized using SuperSignal™ West Pico Plus Chemiluminescent Substrate Kit (Thermo Fisher, 34577). All the antibodies were diluted in PBS with 1% BSA. Primary antibodies used include GAPDH (Santa Cruz, sc-25778), Phospho-p44/42 MAPK (Erk1/2) antibody (CST, 9101), p44/42 MAPK (Erk1/2) antibody (CST, 9017), phospho-SMAD2 antibody (CST, 3108), Smad2 (D43B4) antibody (CST, 5339).
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!