Following the appropriate treatment, cells plated on chamber slides were washed once with 1× PBS and fixed with 4% formaldehyde for 15 min at 23°C. Following fixation, slides were washed with 1× PBS and cells were permeabilized with 0.2% Triton X-100 for 15 min, washed again, and placed in blocking solution (5% normal goat or horse serum; Vector Laboratories, Burlingame, CA) for 30 min. Slides were then incubated in primary antibody for 2 hr at 23°C. Primary antibodies consisted of RXR-γ (1:200; Abcam), myc (1:200; Origene), and phospho-JNK (1:200; Cell Signaling). Following incubation with primary antibody, slides were rinsed 3× with PBS, and incubated with fluorescein isothiocyanate (FITC) (1:500; Vector Laboratories) or Texas Red isothiocyanate (TRITC) (1:500; Vector Laboratories)-conjugated secondary antibodies for 1 h at 23°C in the dark. Sections were again washed 3× with PBS, cover-slipped with an aqueous based mounting medium containing DAPI for nuclear labeling (Vectashield; Vector Laboratories), and visualized with Leica Advanced Widefield imaging system (Leica Microsystems; Buffalo Grove, IL).
Fluorescein isothiocyanate (fitc)
Manufactured by Vector Laboratories
Sourced in United States
FITC (Fluorescein Isothiocyanate) is a fluorescent dye used in various laboratory applications. It is a small molecule that can be conjugated to a wide range of biomolecules, such as antibodies, proteins, and nucleic acids. FITC exhibits green fluorescence when excited by blue or ultraviolet light, making it a useful tool for labeling and detection in experimental techniques.
Automatically generated - may contain errors
Lab products found in correlation
Vectashield, by Vector Laboratories (5 mentions)
Leica advanced widefield imaging system, by Leica Microsystems (3 mentions)
Cot 1 dna, by Roche (3 mentions)
Isis digital image analysis software system, by MetaSystems (3 mentions)
Lsm 710 confocal microscope, by Zeiss (3 mentions)
Normal horse or goat serum, by Vector Laboratories (2 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (2 mentions)
Rhodamine, by Roche (2 mentions)
Texas red, by Vector Laboratories (2 mentions)
Bx61 microscope, by Olympus (2 mentions)
Paraformaldehyde (pfa), by Merck Group (2 mentions)
Vectashield mounting medium with dapi, by Vector Laboratories (2 mentions)
Lsm 710, by Zeiss (2 mentions)
Rxr γ, by Abcam (1 mentions)
Phospho jnk, by Cell Signaling Technology (1 mentions)
Cathepsin b, by Merck Group (1 mentions)
Normal goat serum (ngs), by Vector Laboratories (1 mentions)
Eclipse e400, by Vector Laboratories (1 mentions)
Synaptophysin, by Abcam (1 mentions)
Iba 1, by Abcam (1 mentions)
25 protocols using fluorescein isothiocyanate (fitc)
1
Immunofluorescent Analysis of Protein Localization
2
Immunohistochemical Analysis of Basal Ganglia
3
Comparative Genomic Hybridization Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
CGH was performed as previously described [27 (link)]. Briefly, DNA from UM-SCC1 cells and reference DNA from the blood of a healthy donor were obtained using standard phenol/chloroform extraction, labeled with biotin and digoxigenin by nick translation according to the manufacturer's protocol (Roche Diagnostics, Mannheim, Germany). Hybridization was performed together with COT-1 DNA (Roche Diagnostics) to normal chromosome metaphase spreads from peripheral blood lymphocytes prepared using the following standard procedures. Post-hybridization washes were performed with 50% formamide/2× standard saline citrate (SSC), 2× SSC and 0.1 × SSC at 45°C. DNA was visualized with fluorescein-isothiocyanate (FITC, Vector Laboratories, Burlingame, CA) and rhodamine (Roche Diagnostics), respectively, and counterstained with a DAPI (4, 6-diamidino-2-phenylindole) anti-fade solution (Vector Laboratories). Fluorescence images were captured using a fluorescence microscope Olympus BX 61 and image processing was performed using the ISIS digital image analysis software system (MetaSystems, Altlussheim, Germany). Average ratio profiles were determined from the analysis of 12–15 metaphases.
4
Immunocytochemical Analysis of Cellular Markers
5
Immunocytochemical Analysis of Neural Cell Markers
6
Immunohistochemical Analysis of JEV Infected Mouse Brains
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
JEV infected and age matched control BALB/c mice in sets of three were perfused transcardially with 1× PBS and whole brains isolated were fixed with 4% paraformaldehyde (PFA) in 1× PBS for 24 h at 4°C and subsequently kept in 30% sucrose for additional 24–48 h at 4°C. The brains were then processed for cryostat sectioning and the sections were stained for selected proteins and, a neuronal marker NeuN. Briefly, the cryostat sections were washed with 1× PBS and processed for antigen retrieval by incubating at 70°C for 1 h in antigen unmasking solution (Vector laboratories). The sections were then washed with 1× PBS and blocked for 1.5 h with 5% BSA in 1× PBS. Neurons were labeled with mouse anti-NeuN antibody (1∶500; Chemicon, USA), along with mouse/rabbit specific antibodies (1∶500; Abcam Cambridge, UK) by incubating the sections overnight at 4°C. After five washes with 1× PBS, the sections were incubated with horse anti-mouse Fluorescein Isothiocyanate (FITC, 1∶250; Vector Laboratories) for NeuN and goat anti-rabbit Alexa Fluor 594 (1∶1000; Molecular Probes, Oregon, USA) for selected antibodies for 1 h. The slides were then mounted with mounting medium containing DAPI (Vector laboratories). The images were captured with Zeiss Axioplan 2 upright digital microscope (40× magnification; Zeiss, Germany).
7
Microglial Activation and Neuronal Apoptosis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Iba-1 (1:500, wako, USA) staining was performed on different brain region sections. Anti-Rabbit Alexa fluor 488 (1:1000; Molecular Probes, Invitrogen, USA) was probed as the secondary antibody of Iba-1. Fluorescence immunohistochemistry was performed for Mouse anti-Iba-1(1: 300, Chemicon, USA) for activated microglia which were then double stained with rabbit anti-iNOS (1:1000, Chemicon, USA) and rabbit anti-COX-2 (1:100, Cell Signaling Tech., Boston, USA), respectively. The corresponding secondary antibodies were used goat anti-mouse Alexa Fluor 594 (1:1000; Molecular Probes, OR) for Iba-1, goat anti-rabbit Fluorescein Isothiocyanate (FITC, 1:200; Vector Laboratories, USA) for both iNOS and COX-2. Double staining with NeuN: sections were deparaffinized, subjected to heat-induced epitope retrieval and incubated with NeuN antibody. After NeuN detection with an Alexa 488-labelled secondary antibody, sections were processed for TUNEL staining. Cell nuclei were stained with DAPI.
8
Immunofluorescence Staining of CHPV and N-Protein
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
CHPV protein staining and LC3B (Abcam, United States) was performed on different brain sections. Anti-Rabbit Alexa fluor 488 (1:1000; Molecular Probes, Invitrogen, United States) was probed with the secondary antibody of CHPV protein. The corresponding secondary antibodies Fluorescein Isothiocyanate (FITC, 1:200; Vector Laboratories, United States) for N-protein were used.
9
Retinal Tissue Immunocytochemistry Protocols
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The retinal tissues were processed as whole mounts or 50 μm vertical sections, which were cut using a Vibratome 3000 Plus Sectioning System (St. Louis, Missouri, USA). The immunocytochemical methods have been described in detail previously [14 (link)]. The primary antibodies used were monoclonal anti-PKC (1:100, Amersham, Arlington Heights, Illinois, USA) and polyclonal anti-α1c L-type voltage-gated Ca2+ channel (1:200–1000, Chemicon, Temecula, California, USA). Secondary antibodies used were fluorescein isothiocyanate (FITC)-conjugated horse anti-mouse IgG (1:50–250, Vector Laboratories, Burlingame, California, USA) and Cy5-conjugated goat anti-rabbit IgG (1:50, Jackson ImmunoResearch, West Grove, Pennsylvania, USA). Following the immunocytochemical procedures, the tissues were mounted in Vectashield mounting medium (Vector Laboratories).
10
Immunocytochemistry Protocol for Cellular Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were fixed in 10% (v/v) formaldehyde/PBS at 4°C for 1 hour. Cells were permeabilized with 0.1% (v/v) Triton X-100 in PBS for 10 min at room temperature and blocked with 1% (w/v) bovine serum albumin in PBS with 0.1% (v/v) Tween 20 (PBST) for 1 hour at room temperature. Following blocking, the relevant primary antibodies (Table 2 ) were incubated with cells in blocking buffer overnight at 4°C. Cells were washed three times in PBST and incubated with biotinylated secondary antibodies in blocking buffer (1:50; Vector Laboratories) for 1 hour at room temperature. Cells were again washed three times in PBST and incubated with fluorescein isothiocyanate– or Texas Red–conjugated streptavidin in blocking buffer (1:50; Vector Laboratories). Where appropriate, cell F-actin was labeled through 1-hour incubation at room temperature with rhodamine-conjugated phalloidin (1:1000 in blocking buffer). Nuclei were stained using VECTASHIELD mountant with 4′,6-diamidino-2-phenylindole nuclear stain (Vector Laboratories).
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!