Immunocytochemical analyses were performed on 10 μm frozen tissue sections. Sections were incubated with 10% normal goat serum (Thermo-Fisher) for 1 h, then overnight with primary antibodies to SUV39H2 (see above) or pan cytokeratin (F3418, Sigma Aldrich). Blastocysts fixed in 4% paraformaldehyde, washed with phosphate buffered saline (PBS , pH 7.4), and permeabilized in PBS containing 0.25% Triton X100. Following a blocking step in 10% normal goat serum for 30 min, blastocysts were incubated with the designated primary antibodies to caudal type homeobox 2 (CDX2 , ab76541, Abcam, Cambridge, MA) or POU domain, class 5, transcription factor 1 (POU5F1 , also called OCT4 , C-10, Santa Cruz Biotechnologies, Santa Cruz, CA) overnight at 4°C. After washing with PBS, sections were incubated for 2 h with corresponding secondary antibodies: Alexa488-conjugated goat-anti-mouse immunoglobulin G (IgG , A32723, Thermo-Fisher) or Alexa 568-conjugated goat-anti-rabbit IgG (A11011, Thermo-Fisher). Nuclei were visualized with 4’,6-diamidino-2-phenylindole (DAPI , Molecular Probes, Carlsbad, CA). Fluorescence images were captured using a Leica DMI 4000 microscope equipped with a charge-coupled device camera (Leica Microsystems GMbH, Welzlar, Germany).
F3418
Manufactured by Merck Group
The F3418 is a laboratory equipment product manufactured by Merck Group. It is designed to perform specific functions in a laboratory setting. The core function of this product is to facilitate certain tasks or procedures, but further details about its intended use or application are not provided in this factual and unbiased description.
Automatically generated - may contain errors
Lab products found in correlation
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (2 mentions)
Bovine serum albumin (bsa), by Merck Group (2 mentions)
Normal goat serum (ngs), by Thermo Fisher Scientific (1 mentions)
Ab76541, by Abcam (1 mentions)
Normal goat serum (ngs), by Abcam (1 mentions)
Oct4 c 10, by Santa Cruz Biotechnology (1 mentions)
Alexa 568 conjugated goat anti rabbit igg, by Thermo Fisher Scientific (1 mentions)
Dmi4000 microscope, by Leica Microsystems (1 mentions)
A32723, by Thermo Fisher Scientific (1 mentions)
Vectashield h 1200, by Vector Laboratories (1 mentions)
8 chamber slide, by Thermo Fisher Scientific (1 mentions)
Fitc conjugated anti cytokeratin antibody, by Merck Group (1 mentions)
V6630, by Merck Group (1 mentions)
A11029, by Thermo Fisher Scientific (1 mentions)
A11031, by Thermo Fisher Scientific (1 mentions)
Ds fi3, by Nikon (1 mentions)
Alexa 488 conjugated goat anti mouse secondary antibody, by Thermo Fisher Scientific (1 mentions)
A11001, by Thermo Fisher Scientific (1 mentions)
80i upright microscope, by Nikon (1 mentions)
Alexa 568 conjugated goat anti rabbit secondary antibody, by Thermo Fisher Scientific (1 mentions)
6 protocols using f3418
1
Immunocytochemical Analysis of Embryonic Markers
2
Visualizing Murine Endometrial Epithelial Cells
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Mouse endometrial epithelial cells were seeded at a density of 2×106 cells/well into six-well plates, with each well containing sterile coverslips. Following 24 h growth, 4% paraformaldehyde in PBS was used to fix cells for 20 min at 37°C. Cells were washed with PBS and subsequently permeabilized with PBS containing 1% bovine serum albumin (BSA; Sigma-Aldrich; Merck KGaA) and 0.2% Triton X-100 for 20 min at room temperature. Cells were blocked for 2 h at room temperature with 1% BSA in PBS followed by washing with PBS. Cells were incubated with anti-cytokeratin pan-fluorescein isothiocyanate (1:1,000; F3418; Sigma-Aldrich; Merck KGaA) overnight at 4°C. Cells were washed with PBS three times and nuclei were stained with DAPI (1 µg/ml) for 1 min at room temperature. Subsequently 70% glycerol was utilized to place coverslips on the slides and cells were imaged using a laser scanning confocal microscope (magnification, ×100).
3
Immunostaining of DC2 Cells
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For immunostaining, the DC2 cells were cultured on an 8-chamber slide (Lab-Tek, Nunc Inc. Naperville, Illinois), then washed twice with PBS and fixed in precooled (−20°C) 100% methanol solution for 2 min. Cells were then washed again twice with PBS and air-dried prior to storage at −20°C until use. The fixed cells were serially hydrated for 15 min with PBS, then treated with PBS pH 7.2 containing 1% SDS for 4 min at room temperature. The slides were washed twice with PBS for 5 min then incubated with PBS containing 1% (w/v) BSA (Sigma, St-Louis, MO) for 15 min. The fixed cells were incubated with FITC-conjugated anti-cytokeratin antibody (1:100, F3418, Sigma, St-Louis, MO) overnight at 4°C in a moist chamber. Cells were then washed twice in high salt PBS (PBS containing 2.7% (w/v) NaCl) for 5 min and then washed twice with PBS for 5 min. DAPI mounting medium (VectaShield, H-1200, Vector Laboratories, Burlingame, CA) was used prior to coverslip application and immunofluorescence-stained samples were examined with a Zeiss Axioskop 2 epifluorescence microscope (Carl Zeiss Canada).
4
Immunohistochemical Analyses of Vimentin and Cytokeratin
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Immunohistochemical analyses were performed on 10-μm-thick frozen tissue sections using indirect immunofluorescence. Primary antibodies to vimentin (1:1000; V6630, Sigma-Aldrich) and pan-cytokeratin (1:1000; F3418, Sigma-Aldrich) were used in the analyses. Goat anti-mouse IgG conjugated with Alexa 488 (1:1000; A11029, Thermo Fisher Scientific) and goat anti-mouse IgG conjugated with Alexa 568 (1:400; A11031, Thermo Fisher Scientific) were used to detect primary antibodies. Fluoromount-G™, with 4′6-diamidino-2-phenylindole (DAPI; 00-4959-52, Thermo Fisher Scientific), was used to visualize nuclei and as mounting medium. Processed tissue sections were examined and images were captured with a Nikon Eclipse 80i upright microscope equipped with a charge-coupled device camera (Nikon DS-Fi3).
5
Immunofluorescent Analysis of Placental and Trophoblast Cells
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Frozen placental tissues were sectioned at a thickness of 10 µm with a cryostat. Immunohistochemical analysis was performed on placental sections by immunofluorescence detection using a pan-cytokeratin primary antibody (1:250, F3418; Sigma-Aldrich) and Alexa 488-conjugated goat-antimouse secondary antibody (1:200, A11001; ThermoFisher). Nuclei were visualized with 4’,6-diamidino-2-phenylindole (Molecular Probes, Eugene, Oregon, USA). Rat TS cells were fixed with 4% paraformaldehyde (Sigma-Aldrich) for 20 min at room temperature. Immunocytochemical analysis was performed by immunofluorescence detection using a primary antibody against prolactin family 8, subfamily A, member 5 (PRL8A5, 1:20016 (link)) followed by Alexa 568-conjugated goat-anti-rabbit secondary antibody (1:200, A11001; ThermoFisher). Images were captured on a Nikon 80i upright microscope (Nikon, Melville, New York, USA) with a Photometrics CoolSNAP-ES monochrome camera (Roper, Sarasota, Florida, USA).
6
Immunostaining of Developing Urinary Tract
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