The PC12 cells were seeded on glass slides, cultured and treated as previously described. At the end of each time interval, the cells were fixed in Shandon™ Glyo-Fixx™ for 30 min, washed 3 times in PBS 1X, incubated 30 min at 37 °C with rabbit serum in PBS 1X and incubated overnight at 4 °C with anti-cytochrome c primary antibody (sc13560, Santa Cruz Biotechnology, Dallas, Texas, USA). The next day, the samples were washed twice with PBS 1X and incubated 1 h in the dark with a TRITC-conjugated secondary antibody (Sigma Aldrich, Milan, Italy). After washing with PBS 1X, the cell nuclei were stained in blue by DAPI and the slides were mounted as previously described. The images were acquired at 40X magnification.
Tritc conjugated secondary antibody
Manufactured by Merck Group
Sourced in Italy, United States
TRITC-conjugated secondary antibody is a fluorescently labeled antibody used in various immunodetection techniques. It binds to the primary antibody, allowing for the visualization of target proteins or other molecules in a sample. The TRITC (Tetramethylrhodamine) fluorescent dye emits an orange-red color when excited, enabling detection and localization of the labeled target.
Automatically generated - may contain errors
Lab products found in correlation
Type 1collagen, by Thermo Fisher Scientific (1 mentions)
Axio imager m2, by Zeiss (1 mentions)
Anti fibronectin, by Merck Group (1 mentions)
Axiovision, by Zeiss (1 mentions)
Dabco, by Merck Group (1 mentions)
Monoclonal anti brdu antibody clone bu 33, by Merck Group (1 mentions)
Axiovision software, by Zeiss (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Vector Laboratories (1 mentions)
Eclipse 80i epifluorescence microscope, by Nikon (1 mentions)
Lypla1, by Abcam (1 mentions)
Sec62, by Merck Group (1 mentions)
Eif2ak2, by Abcam (1 mentions)
Vps28, by Merck Group (1 mentions)
Lsm 510 meta microscope, by Zeiss (1 mentions)
β actin, by Santa Cruz Biotechnology (1 mentions)
H6908, by Merck Group (1 mentions)
F1804, by Merck Group (1 mentions)
8 protocols using tritc conjugated secondary antibody
1
Cytochrome c Immunofluorescence in PC12 Cells
2
Immunofluorescence Imaging of Cardiac Fibrosis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Samples of cardiac tissue, from both noninfected and T. cruzi-infected mice, were frozen in Tissue-Tek O.C.T Compound (Sakura, Zoeterwoude, The Netherlands) and stored in liquid nitrogen. Heart cryosections were fixed in cold acetone, air-dried and stored at -20 °C. Sections were washed in PBS and then blocked with PBS containing 4% serum albumin bovine (BSA; PBS + BSA) before immunostaining. The samples were incubated overnight at 4 °C with anti-fibronectin (1:600, Sigma Chemical Co., St Louis - USA) or type 1 Collagen (1:400; Life Technologies Co., Carlsbad, CA - USA) antibodies followed by washing with PBS + BSA. The antigen-antibody complex was detected by incubation for 1 h at 37 °C with TRITC-conjugated secondary antibody (1:400, Sigma). After washing, the sections were incubated with 4′,6-diamidino-2-phenylindole (DAPI), a DNA staining, and mounted with the anti-fading DABCO® (Sigma). Images were acquired under a Zeiss Axio Imager M2 fluorescence microscope using AxioVision (Zeiss) digital image processing software.
For the type I collagen bioimage analysis, at least five different fields were captured from each group (uninfected and infected), and fluorescence images were segmented and the mean intensity measured using Knime workflow.33 (link)
For the type I collagen bioimage analysis, at least five different fields were captured from each group (uninfected and infected), and fluorescence images were segmented and the mean intensity measured using Knime workflow.33 (link)
3
Immunofluorescent Localization of p-ERK1/2 and MMP9
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were grown on coverslips and fixed in 4% paraformaldehyde at room temperature for 10 min. After PBS washing, the cells were blocked with 3% BSA at 37 °C for 30 min and incubated at 4 °C with anti-p-ERK1/2 or MMP9 overnight, and then probed with a tetramethylrhodamine isothiocyanate (TRITC)-conjugated secondary antibody (Sigma, USA) at 37 °C for 2 h. Subsequently, cells were stained with DAPI and observed under a fluorescence microscope.
4
Quantification of Cell Proliferation by BrdU Incorporation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Bromodeoxyuridine (5-bromo-2′-deoxyuridine; BrdU) is known to be incorporated into the newly synthesized DNA of replicating cells during the S phase of the cell cycle. To identify actively replicating cells, and thereby assess cellular proliferation, BrdU was applied to cells at days 7, 10, and 12 of culture. To visualize BrDU, cultures were incubated for 2 h with a monoclonal anti-BrdU antibody (clone BU 33; Sigma). After fixation with methanol for 10 min, the cells were treated with 1 N HCl for 1 h at room temperature before incubation with a TRITC-conjugated secondary antibody (Sigma) for 1 h in a dark room. Finally, the cells were stained with 1 mg/mL 4′,6-diamidino-2-phenylindole (DAPI; Vectashield; Vector Laboratories, Burlingame, CA). Immunofluorescence was observed using a confocal microscope (Carl Zeiss, Jena, Germany) and images were acquired using AxioVision software (Carl Zeiss). Images (n = 3 for each experimental conditions) were converted to binary and analyzed using ImageJ software (version 1.44; National Institutes of Health, Bethesda, MD).
5
Immunofluorescent Staining of LC3 in HK-2 Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The HK-2 cells were fixed with cold acetone. 3% bovine serum albumin (BSA) was used to block antigen and the cells were incubated with the primary antibody against LC3 (1:200) at 4 °C overnight. Then, we washed the sections with PBS and incubated them with a tetramethylrhodamine isothiocyanate (TRITC)-conjugated secondary antibody (Sigma-Aldrich) in the dark for 1 h. After being washed with PBS, cells were visualized with a Nikon Eclipse 80i Epi-fluorescence microscope.
6
FMDV Infection Dynamics in BHK-21 Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Coverslip-adhered BHK-21 cell monolayers were infected with FMDV at an MOI of 1. The plates were inoculated at 37°C over 1 h followed by replacement with fresh growth media, and were incubated at 37°C for another 5 h. The cells were fixed with 4% PFA and treated with 0.01% Triton X-100, with the following antibodies applied: DARS (Santa Cruz Biotechnology, Inc., USA), LYPLA1 (Abcam, UK), SEC62 (Sigma-Aldrich, USA), EIF2AK2 (Abcam, UK), EVI5 (Sigma-Aldrich, USA),VPS28 (Sigma-Aldrich, USA), or β-actin (Santa Cruz Biotechnology, Inc., USA). FMDV proteins were detected by pig anti-FMDV Asia 1 VLPs serum (primary antibody, 1:100) recognized by a TRITC-conjugated secondary antibody (Sigma). DAPI was used as counterstain to allow visualization of cell nuclei. Immunofluorescence confocal microscopy images were captured on an LSM510 META microscope (Carl Zeiss, Ltd.).
7
Immunofluorescence Imaging of β-Catenin
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were grown on coverslips and fixed in 4% paraformaldehyde at room temperature for 10 min. After PBS washing, the cells were blocked with 10% goat serum at 37°C for 30 min, and incubated at 4°C with anti‐β‐catenin overnight, and then probed with a tetramethyl rhodamine isothiocyanate (TRITC)‐conjugated secondary antibody (Sigma) at 37°C for 2 h. Subsequently, cells were stained with DAPI and observed under a fluorescence microscope.
8
Cotransfection and Immunofluorescence Imaging
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
DF-1 cells were cotransfected with pCAH-VP4 and pCAF-CypA. Single transfection with pCAH-VP4 or pCAF-CypA was performed as controls. After 48 h, the cells were fixed with 4% paraformaldehyde for 30 min at 37°C, permeabilized with 0.1% Triton X-100 for 10 min, and blocked with 3% bovine serum albumin (BSA). Anti-HA rabbit polyclonal antibody (H6908, Sigma) and anti-Flag mouse mAb (F1804, Sigma) were incubated with the cells for 1 h. After washing five times with PBS, the cells were incubated with the FITC- or TRITC-conjugated secondary antibody (Sigma) for 1 h at 37°C. The cells were then stained with DAPI for 10 min at 37°C and examined using a Leica SP2 Confocal system (Leica Microsystems, Wetzlar, Germany).
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!