Cells grown on glass coverslips were washed with PBS, fixed with 4% paraformaldehyde in cytoskeleton buffer (CB; 20 mM Pipes, pH 6.8, 300 mM NaCl, 10 mM EGTA, 10 mM glucose, and 10 mM MgCl2), and permeabilized with 0.05% saponin in blocking buffer (CB supplemented with 10% goat serum [G6767; Sigma-Aldrich] and 5% BSA [8076.5; Roth]). Slides were subsequently incubated with primary antibodies in blocking buffer for 1.5 h at RT, washed six times with CB, incubated light-protected with secondary antibody in blocking buffer for 1 h at RT, and washed six times with CB. Coverslips were then mounted in Mowiol. Epifluorescent images were acquired using a 63× oil immersion objective (NA 1.4) and the Axio Imager M1 microscope (Carl Zeiss) equipped with a SPOT Xplorer (Visitron Systems) camera at RT. Acquisition was controlled by the VisiView software (Visitron Systems GmbH). Confocal stacks and single confocal planes were acquired using a Laser Scanning Confocal Microscope (SP5; Leica) equipped with a 63× oil objective (NA 1.4) at RT. Acquisition was controlled by the LAS AF software (Leica). Confocal images were deconvoluted with Huygens Professional Deconvolution and Analysis Software (Scientific Volume Imaging) by applying Classic Maximum Likelihood Estimation, reformatted to TIFF using ImageJ, and adjusted for brightness and contrast using Photoshop CS2.
G6767
Manufactured by Merck Group
Sourced in United Kingdom
G6767 is a laboratory centrifuge designed for general-purpose applications. It is capable of separating components of a liquid mixture based on their different densities. The device can accommodate various sample volumes and rotation speeds to meet the needs of diverse research and testing requirements.
Automatically generated - may contain errors
Lab products found in correlation
Bovine serum albumin (bsa), by Carl Roth (1 mentions)
Las af software, by Leica (1 mentions)
Spot xplorer, by Visitron (1 mentions)
Axio imager m1 microscope, by Zeiss (1 mentions)
Alexa488 conjugated anti mouse, by Thermo Fisher Scientific (1 mentions)
Rnase a, by Merck Group (1 mentions)
A21236, by Thermo Fisher Scientific (1 mentions)
A11126, by Thermo Fisher Scientific (1 mentions)
Prolong glass, by Thermo Fisher Scientific (1 mentions)
D8537, by Merck Group (1 mentions)
T6664, by Merck Group (1 mentions)
Vectashield mounting medium, by Eurobio Scientific (1 mentions)
Alexa 647, by Thermo Fisher Scientific (1 mentions)
4 6 diamidin 2 phenylindol dapi, by Merck Group (1 mentions)
Alexa 488, by Thermo Fisher Scientific (1 mentions)
Dmi4000 microscope, by Leica (1 mentions)
Dako fluorescence mounting medium, by Agilent Technologies (1 mentions)
7 protocols using g6767
1
Immunofluorescence Staining on Coverslips
2
Immunostaining of Mouse Embryos
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Embryos were harvested in PBS, pinned to a silicon‐coated Petri dish and excess tissue trimmed with a blade. For GM130 (1:100, mouse, BD Biosciences #610822) and N‐cadherin (1:100, mouse, BD Biosciences, #610921) antibody staining, fixation was in 4% Paraformaldehyde (PFA) at room temperature for 15 min. For PKCζ (1:100, rabbit, SantaCruz, SC216), PAR3 (1:300, rabbit, Upstate, 07330) and ZO1 (1:50, rat, SantaCruz, SC33725) antibody staining, embryos were fixed in 1% PFA for 15 min at 4°C. Three embryos per marker were used except for ZO1 where two embryos were analysed. After fixation, embryos were permeabilized overnight in PBST (1% Triton‐X100 in PBS) with 0.02% thimerosal and blocked for a day at 4°C with 0.1% BSA (Sigma, A3803‐50G) with 5% heat‐inactivated goat serum (Sigma, G6767) in PBST. Then, embryos were incubated in blocking solution with the desired primary antibody and washed several times overnight in PBST. They were then incubated in the dark for 1 day in blocking solution with the appropriate secondary antibody: Alexa 488‐conjugated anti‐mouse (Life Technologies, A21202), anti‐rabbit (Life Technologies, A11008) or anti‐rat (Life Technologies, A11006) together with 1:2000 ToPro3 nuclear stain (Molecular probes, T3605) and 10 μg/mL RNase A (Sigma, R6513). Finally, embryos were cleared and mounted for imaging (see Supplementary Methods ).
3
Immunofluorescent Staining of Tubulin in BS-C-1 Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
BS-C-1 cells (CCL-26, American Type Culture Collection) were plated on custom, glass-bottom dishes (Catalog # PG35G-10C-NON, MatTek Corporation) fitted with high-performance Zeiss coverglasses (Catalog # 474030-9000-00, Thickness 1.5) at a density of 20,000 cells and incubated for 16–24 h. The coverglasses were cleaned by sonicating them sequentially in 50% HPLC grade ethanol, 1 mM HCL in 50% HPLC grade ethanol, and 1 M KOH in 50% HPLC grade ethanol for 20 min each with extensive washing using MilliQ water after every sonication. The cells were fixed using 3.4% paraformaldehyde (Electron Microscopy Sciences) in PBS for 10 min at 37 °C. The fixed cells were then permeabilized using 0.1% Triton X-100 in PBS for 10 min at room temperature. Cells were then preblocked with 5% BSA (Catalog # BP1600, Fisher Scientific), incubated with mouse α-tubulin antibody (Catalog # A11126, Thermo Fisher Scientific, diluted 200-fold in 1% BSA/PBS) for 30 min at room temperature, and treated with goat serum (Catalog # G6767, Sigma, diluted 50-fold in 1% BSA/PBS). Bound primary antibody was counter-stained with Alexa 647-labeled goat anti-mouse IgG (Catalog # A-21236, Thermo Fisher Scientific, diluted 750-fold in 1% BSA/PBS) for 30 min at room temperature.
4
Immunofluorescent Staining of Cultured Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For immunofluorescent staining the cells were seeded on #1.5 glass coverslips. At
approximately 80% confluence the cells were subjected to treatment. The cells were then fixed using 4% formaldehyde (ThermoFisher Scientific, J19943.K2) at 37°C for 20 min. The cells were permeabilized with methanol at room temperature for 5 min. The permeabilized cells were blocked with 3% pre-immune goat serum (Sigma-Aldrich, G6767) in phosphate-buffered saline (PBS; Sigma-Aldrich, D8537) for 1 h at room temperature before overnight incubation at 4°C with primary antibodies diluted in PBS with 1% goat serum. Cells were then washed and incubated for 1 h with Alexa Fluor-coupled secondary antibodies diluted 1:500 in PBS supplemented with 1% goat serum. After a final wash in PBS the coverslips were mounted on glass slides using Prolong Glass (Invitrogen, P36980).
approximately 80% confluence the cells were subjected to treatment. The cells were then fixed using 4% formaldehyde (ThermoFisher Scientific, J19943.K2) at 37°C for 20 min. The cells were permeabilized with methanol at room temperature for 5 min. The permeabilized cells were blocked with 3% pre-immune goat serum (Sigma-Aldrich, G6767) in phosphate-buffered saline (PBS; Sigma-Aldrich, D8537) for 1 h at room temperature before overnight incubation at 4°C with primary antibodies diluted in PBS with 1% goat serum. Cells were then washed and incubated for 1 h with Alexa Fluor-coupled secondary antibodies diluted 1:500 in PBS supplemented with 1% goat serum. After a final wash in PBS the coverslips were mounted on glass slides using Prolong Glass (Invitrogen, P36980).
5
Immunofluorescence Staining Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were washed with Tris-Buffered Saline (TBS) (TBS; T6664; Sigma-Aldrich, Dorset, UK) for 2 minutes prior to fixation in 4% formaldehyde (28908, Thermo Scientific, Massachusetts, USA) at 4C for 1 hour. Cells were then washed with TBS and permeabilized with 0.1% Triton X 100 (85111, ThermoFisher, Massachusetts, USA) for 30 minutes. Following a further TBS wash, samples were blocked with 5% goat serum (G6767; Sigma-Aldrich, Dorset, UK) for 1 hour. Primary antibody was then added and incubated at 4C overnight (Primary antibodies listed in Table 1) after which, cells were washed in TBS on a shaker for approximately 20 minutes and then washed again before adding secondary antibody, Hoescht and phalloidin.
Secondary antibody incubation was at 4C for 4 to 24 hours, depending on antibody, and three further wash steps were performed after incubation and before microscopy.
Secondary antibody incubation was at 4C for 4 to 24 hours, depending on antibody, and three further wash steps were performed after incubation and before microscopy.
6
Immunofluorescence Staining of Cultured Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cultured cells were fixed in 4% PFA for 15 min at room temperature, washed 3 × 10 min in 1X PBS and incubated in blocking and permeabilization solution for 1 h at room temperature (1× PBS; 0.2% Triton X-100; 10% normal goat serum, Sigma, G6767). Primary antibody was diluted in blocking and permeabilization and incubated over night at 4 °C. Primary antibodies and dilutions are indicated in Supplementary Table 4 . Following 3 × 10 min washes in 1× PBS, cells were incubated with secondary antibody diluted in the blocking and permeabilization solution for 1 h at room temperature. Excess antibody was washed 3 × 10 min in 1× PBS, and incubated with 0.0002% DAPI (Sigma, 10236276001) for 15 min at room temperature. Stained cells were finally washed 3 × 10 min in 1× PBS and mounted with Vectashield® Mounting Medium (Eurobio Scientific, H-100) prior to observation. Images were acquired with a laser scanning confocal microscope (SP8, Leica), using a 20×/0.8NA or 60×/1.4NA objective and Leica Application Suite X software.
7
Immunohistochemistry of Tissue Sections
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Tissues were fixed in 4% PFA for two hours and incubated in 20% sucrose overnight at 4 °C. Tissues were then embedded in Tissue-Tek O.C.T compound (Sakura), snap-frozen and stored at -80 °C. 7 μm thick tissue sections were cut, dried for 30 min, rehydrated in PBS, permeabilized with 0.2% Triton X-100 in PBS for 20 min and blocked with 5% normal goat serum (NGS; Sigma-Aldrich; G6767) in PBS for one hour. Next, sections were incubated with primary antibody overnight at 4 °C followed by 1 h incubation with secondary antibody at room temperature. All antibodies were diluted in 5% NGS in PBS. The following primary antibodies were used: guinea pig-anti-CK8/ 18 (Fitzgerald Industries; 20R-CP004; 1:50), rabbit-anti-CK14 (Thermo Scientific; RB-9020; 1:50), rat-anti-E-cadherin (clone ECCD-2; Invitrogen; 13-1900; 1:400), rabbit-anti-PDGFRβ (clone Y92; abcam; ab32570; 1:100). Secondary antibodies directed against the species of the primary antibody were coupled to Alexa 488 or Alexa 647 (Invitrogen; 1:400). Nuclei were stained with 4′,6-Diamidin-2-phenylindol (DAPI; Sigma-Aldrich; 1:5000) for 10 min at room temperature. The sections were mounted with Dako fluorescence mounting medium (Agilent). Images were acquired with a Leica DMI 4000 microscope and processed with ImageJ.
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!