SVF cells were fixed in 4% PFA for 15 min at room temperature. Cells were then washed with PBS prior to a 10 min incubation in permeabilization buffer (0.2% Triton X-100, 1% BSA in PBS). Next, the cells were washed with PBS and blocked for 1 h in PBS/1% BSA solution. After blocking, cells were incubated with anti-Lamin B1 primary antibody (1:100, 12987-1-AP, Proteintech, Rosemont, IL) or anti-p21 primary antibody (1:40, 14-6715-81, Thermo Fisher Scientific) in PBS/1% BSA overnight at 4 °C. The next day, cells were washed with PBS and incubated for 1 h at room temperature with Alexa Fluor 647-conjugated anti-rabbit secondary antibody (A-21244, Thermo Fisher Scientific) diluted 1:200 in PBS/1% BSA. Stained cells were then washed with PBS and analyzed by flow cytometry or ImageStreamX.
Alexa fluor 647 conjugated anti rabbit secondary antibody
Manufactured by Thermo Fisher Scientific
Alexa Fluor 647-conjugated anti-rabbit secondary antibody is a detection reagent used in fluorescence-based applications. It binds to primary antibodies raised in rabbit and is labeled with the Alexa Fluor 647 fluorescent dye, which can be detected using appropriate instrumentation.
Automatically generated - may contain errors
Lab products found in correlation
Anti p21 primary antibody, by Thermo Fisher Scientific (1 mentions)
Trypsin, by Thermo Fisher Scientific (1 mentions)
Facscanto 2, by BD (1 mentions)
Anti dclk1, by Abcam (1 mentions)
Zen blue 2, by Zeiss (1 mentions)
Axio scan z1 slide scanner, by Zeiss (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions)
Alexa fluor 488 conjugated anti mouse secondary antibody, by Thermo Fisher Scientific (1 mentions)
Facsverse, by BD (1 mentions)
Cd133, by BD (1 mentions)
4 protocols using alexa fluor 647 conjugated anti rabbit secondary antibody
1
Immunofluorescent Staining of Senescent Cells
2
Quantification of NMDA-R Expression in Macrophages
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
PMs were cultured in DMEM/F12 with 10% FBS alone or in the presence of 20% L929 cell-conditioned medium or 10 nM CSF-1 for up to 5 days. PMs and BMDMs were suspended using 0.5% Trypsin (Invitrogen). Non-specific antibody binding was blocked by incubation with 2% BSA in PBS. The cells were then treated with NMDA-R NR1 subunit-specific antibody (Invitrogen, cat. PA3-102), diluted 1:100 for 40 min. After washing to remove unbound antibody, the cells were incubated with Alexa Fluor 647-conjugated anti-rabbit secondary antibody (ThermoFisher, 1:600) for 30 min. Unbound antibody was removed by washing. All incubations were performed with live cells at 4°C so that antibody-binding was limited to cell surface epitopes. At least 10,000 cells per sample were analyzed using a BD FACS Canto II (BD Biosciences). Flow histogram overlays were prepared using FlowJo software (FlowJo, LLC).
3
Quantification of Intestinal Tuft Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Small intestines were collected, formalin fixed, and paraffin embedded. Paraffin sections (5 µm) were deparaffinized in xylene and rehydrated in an ethanol gradient. Antigen retrieval was performed in 10 mM sodium citrate buffer, pH 6.0, at 95°C for 20 min. Sections were stained with rabbit polyclonal anti-DCLK1 (1:1,000; Abcam) overnight at 4°C, followed by staining with Alexa Fluor 647–conjugated anti–rabbit secondary antibody (1:4,000; Molecular Probes), and counterstaining with DAPI (1:10,000; Sigma-Aldrich). Images were acquired with the Axio Scan Z.1 Slide Scanner (ZEISS). Quantification was performed blind by counting the total number of tuft cells per crypt-villus with the Zen Blue 2 software (ZEISS).
4
Immunophenotyping of Harvested Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Harvested cells were incubated in blocking solution with primary antibodies against FAP (AbCam), CD90 (Novus Biologicals), CD133 (BD Biosciences), and CD44 (BD Biosciences) on ice. Cells were then further incubated with Alexa Fluor 488-conjugated anti-mouse secondary antibody (Molecular Probes) or Alexa Fluor 647-conjugated anti-rabbit secondary antibody (Molecular Probes) on ice and in the dark. Flow cytometry was performed using FACSVerse (BD Biosciences) immediately after washing with PBS.
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!