For surface staining and cell sorting, single cell suspensions from spleen or bone marrow were stained 15 min on ice with antibodies specific to murine B220, CD19, c-kit, CD25, CD93 (AA4.1), CD38, CD21, CD23, IgA, Streptavidin APC Conjugate (eBioscience), CD95 (Fas), CD138 (BD Biosciences), IgG1, IgG2A-2B (BD PharMingen), and IgM (m chain specific) (Jackson ImmunoResearch). Dead cells were excluded by DAPI staining (Sigma). Annexin V staining was done according to manufacturer’s protocol (eBioscience). For intracellular staining, cells were surface stained and fixation/permeabilization buffers (eBioscience) protocol was followed. Intracellular antibody used was PTEN (Cell Signaling) followed by FITC conjugated anti-rabbit antibody (Jackson ImmunoResearch). For intracellular phosphoflow staining, cells were surface strained then fixed in 2% PFA for 30 min at RT and permeabilized with 90% methanol for 30–60 min on ice. Cells were washed in PBS then stained for AKT, Phospho-AKT Thr308 and Phospho-AKT Ser473 (Cell Signaling) followed by FITC conjugated anti-rabbit antibody (Jackson ImmunoResearch). Samples were analyzed on an LSR Fortessa cytometer or sorted using a FACS Aria II (BD Biosciences). Data were analyzed with FlowJo software (Tree Star).
Fitc conjugated anti rabbit antibody
Manufactured by Jackson ImmunoResearch
Sourced in United States
The FITC-conjugated anti-rabbit antibody is a laboratory reagent used for the detection and identification of rabbit-derived proteins or antigens. It consists of a fluorescein isothiocyanate (FITC) molecule covalently attached to an antibody specific for rabbit immunoglobulins. This reagent can be used in various immunoassay techniques, such as immunofluorescence microscopy, flow cytometry, and Western blotting, to visualize and localize rabbit-derived targets.
Automatically generated - may contain errors
Lab products found in correlation
Cy3 conjugated anti mouse antibody, by Jackson ImmunoResearch (2 mentions)
Cy3 conjugated anti rabbit antibody, by Jackson ImmunoResearch (1 mentions)
Mouse anti nkx2 1 antibody, by LSBio (1 mentions)
Rabbit anti sox2 antibody, by Novus Biologicals (1 mentions)
Rabbit anti k8 antibody, by LSBio (1 mentions)
Vectashield, by Vector Laboratories (1 mentions)
Rhodopsin, by Abcam (1 mentions)
Cy3 conjugated anti mouse, by Jackson ImmunoResearch (1 mentions)
Uorescent microscope, by Leica camera (1 mentions)
Normal goat serum (ngs), by Merck Group (1 mentions)
Recoverin, by Abcam (1 mentions)
Alexa488 conjugated anti mouse, by Jackson ImmunoResearch (1 mentions)
Prolong gold antifade mounting media, by Thermo Fisher Scientific (1 mentions)
Insulin, by Merck Group (1 mentions)
Hydrocortisone, by Merck Group (1 mentions)
Anti p62 antibody, by Merck Group (1 mentions)
Anti lc3b antibody, by Cell Signaling Technology (1 mentions)
Fluoview microscope, by Olympus (1 mentions)
Dapi staining, by Merck Group (1 mentions)
Lsr fortessa cytometer, by BD (1 mentions)
7 protocols using fitc conjugated anti rabbit antibody
1
Multiparameter Analysis of Immune Cells
2
Immunofluorescence Staining of Respiratory Structures
3
Immunocytochemical Characterization of Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
At determined time, the different groups of RPCs were xed with 4% paraformaldehyde (PFA). After blocking with 10% normal goat serum (Sigma-Aldrich), the cells were incubated with one of the following primary antibodies: Ki67, Nestin, GFAP, β-III-tubulin, Recoverin, Rhodopsin (Abcam), Crx (Omnimabs) 4 °C overnight. Then they were labeled with the corresponding secondary antibodies: Alexa 488-conjugated anti-mouse, Cy3-conjugated anti-mouse or FITC-conjugated anti-rabbit antibodies (Jackson). Finally, cells were counterstained with DAPI nuclear stain and observed by uorescent microscope (Leica, Germany). The positive ratio was calculated with (immunepositive cells/DAPI stained cells in the eld) × 100% by ImageJ software.
4
Immunofluorescence Staining of Keratin 6
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were maintained in F12 (Gibco, 11765), 10% FBS, 1% penicillin/streptomycin, 5 ng/mL EGF, 1 μg/mL hydrocortisone (Sigma, H0888), 5 μg/mL insulin (Sigma, I9278). Cells were plated on glass coverslips, and allowed to attach overnight. For immunofluorescence (IF), cells were fixed with 1:1 methanol:acetone at −20 °C for 10 min and rinsed with 1× PBS. Slides were incubated with 50 μL KRT6 primary antibody 1:200 in 3% bovine serum albumin (BSA; Benchmark, 100-106), 0.01% Tween 20 (BIO-RAD, 170-6531), 1× PBS at 37 °C for 90 min with shaking. After washing with 0.01% Tween 20, 1× PBS, slides were incubated with 50 μL FITC-conjugated anti-rabbit antibody (Jackson ImmunoResearch, 711-095-152) 1:100 in 3% BSA, 0.01% Tween 20, 1× PBS at 37 °C for 30 min with shaking. Cells were stained with DAPI (Invitrogen, P36931) and mounted with ProLong Gold Antifade Mounting Media (Life Technologies, P363930). Slides were imaged using the Olympus 1×51 scope and Metamorph Image program.
5
Quantifying Autophagy Markers LC3B and p62
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were washed in phosphate buffered saline (PBS), prefixed in 4% paraformaldehyde for 15s at room temperature (RT), fixed in ice-cold 100% methanol for 10min at 20°C and washed PBS at RT. Primary antibodies, namely anti-LC3B antibody (Cell Signaling, Lausen, Switzerland, rabbit monoclonal, clone D11, #3686) and anti-p62 antibody (Sigma, Leiden, Netherlands, mouse monoclonal, clone 2C11, #WH0008878M1) were diluted 1:100 and 1:200 in PBS/1% bovine serum albumin (BSA)/0.1% Tween, respectively. Slides were incubated with primary antibodies for 1h at RT. Slides was washed twice with PBS/0.1% Tween and once with PBS only. Secondary antibodies, namely FITC conjugated anti-rabbit antibody (Jackson Immunoresearch, Suffolk United Kingdom, #111-096-045) and Cy3 conjugated anti-mouse antibody (Jackson Immunoresearch, Suffolk, United Kingdom, #115-166-003), were diluted 1:130 in PBS/1%BSA/0.1%Tween. Slides were incubated with secondary antibodies for 1h at RT. Images were taken on Olympus FluoView microscope at 63x objective magnification and adjusted for brightness using ImageJ Software (NIH, Bethesda, MD, United States of America, 1.64r).
6
Cell Cycle Analysis by Flow Cytometry
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
To determine the DNA content and mitotic index by flow cytometry, cells were cultured in 10 cm dishes in the presence of the appropriate inhibitors. Culture media was then collected and the remaining cells harvested by trypsinisation. Cells were then pelleted and washed in PBS before finally being resuspended in 150 μl PBS plus 350 μl 100% ice-cold ethanol. Cells were then stored at −20°C for at least 16 hr. Fixed cells were washed twice in PBS and incubated with rabbit anti-phospho-histone H3 (Ser10)(Millipore) diluted 1:1000 in PBS for 1 hour at 4°C. Following a PBS wash, cells were then incubated with a FITC-conjugated anti-rabbit antibody (Jackson Immunoresearch) for one hour at 4°C. Cells were again washed in PBS and resuspended in 500 μl PBS containing 40 μg/ml propidium iodide, 50 μg/ml RNase A. Samples were then incubated in the dark at room temperature for 30 min before being analyzed on a Cyan ADP flow cytometer using Summit analysis software (Dako).
7
Immunofluorescence Assay for DNA Damage
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The cells were cultured on glass coverslips and fixed with 2% paraformaldehyde (Sigma-Aldrich) in phosphate buffered saline (PBS) at room temperature for 15 min. After washing three times for 3 min with PBS, the fixed cells were permeabilized for 5 min at room temperature with PBS containing 0.5% Triton X-100 and washed again as described above. Blocking was performed with PBS containing 0.01% TritonX-100 and 1% bovine serum albumin (1% BSA/PBS-T) for 60 min at room temperature. The cells were incubated with PBS-T containing primary antibodies (1:200 for phospho-Histone H2AX from Cell Signaling) for at 4 °C for overnight, washed four times for 5 min with PBS-T, and incubated with PBS-T containing FITC-conjugated anti-rabbit antibody (Jackson ImmunoResearch Laboratories, PA, USA) for 1 h, followed by 4′,6-diamidino-2-phenylindole (DAPI; 0.5 μg/ml; Roche) for 10 min in the dark at room temperature. The cells were washed for four times with PBS-T and observed with a fluorescence microscope (ZEISS AXioskop2).
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!