Cells seeded in 12-well plates were washed with ice-cold PBS and fixed immediately with 4% paraformaldehyde for 30 min at room temperature (RT). Then, fixed cells were permeabilized with 0.2% Triton X-100 in phosphate-buffered saline (PBS) for 10 min at RT. Subsequently, cells were blocked in 5% bovine serum albumin (in PBS) for 60 min at RT, followed by incubation with primary antibodies against Flag/HA/Myc overnight at 4°C. After washing cells with PBS 3 times, Alex Fluor 488 (Green)/Fluor 592 (Red) conjugated secondary antibodies (Invitrogen) were added to cells for 1 h at RT. Cell nuclei were stained with DAPI (Invitrogen). Images were captured using Leica fluorescence optical microscope.
Fluorescence optical microscope
Manufactured by Leica
The Fluorescence optical microscope is a specialized instrument used for the visualization and analysis of biological samples. It utilizes the principle of fluorescence, allowing for the observation of specific molecules or structures within a specimen that have been labeled with fluorescent dyes or proteins. The microscope's core function is to provide high-contrast, high-resolution images of these fluorescently-labeled components, enabling researchers to study cellular and subcellular processes, protein interactions, and other biological phenomena.
Automatically generated - may contain errors
6 protocols using fluorescence optical microscope
1
Fluorescent Immunocytochemistry Assay
2
5-Hydroxymethylcytosine Immunofluorescence Staining
3
Immunohistochemical Staining of Skin Sections
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For immunohistochemical staining, the skin sections were heat-fixed at 60 °C for 2 h, deparaffinized in xylene for 30 min, and then rehydrated in 100%, 95%, 90%, 80%, and 70% ethanol for 10 min. After washing with PBS and PBST (PBS containing 0.05% Tween-20) for 5 min each, the sections were incubated with the endogenous peroxidase blocking buffer (Beyotime Biotechnology; P0100A, 100 ml) for 10 min. Next, the sections were washed with PBS for three times, and antigen retrieval was conducted for 10 min by steaming in 10 mM citrate antigen retrieval solution. Further, the sections were blocked with 3% bovine serum albumin (BSA; Sigma, V900933, 100 g) which was diluted by PBS for 1 h, then stained with F4/80, CD86, and CD206 at 4 °C overnight, and washed with PBST and PBS properly. The stained sections were incubated in a hypersensitive HRP-labeled goat anti-mouse/rabbit Ig mixture for 20 min and incubated with the signal enhancer reagent. After washing with PBST and PBS, DAB reagent was added and the sections were further incubated for 120 s. Thereafter, the samples were re-stained with hematoxylin for 60 s. Finally, the sections were dehydrated with 70%, 80%, 90%, 95%, 100% ethanol for 1 min each, with xylene for 10 min, and covered with neutral balsam for imaging and observation under a Leica fluorescence optical microscope.
4
Immunofluorescence Staining for TPI1 Protein
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Cells were washed with cold PBS and fixed with 100% Methanol for 15 min at −20 °C. Then, cells were treated with 0.2% Triton X-100 for cell perforation at room temperature for 10 min, and were incubated with blocking buffer (5% BSA in PBS) for 30 min, followed by incubation at 4 °C overnight with the primary antibody against TPI1, and Alex Fluor 488 (Green) conjugated secondary antibody (Invitrogen) at room temperature for 1 h. Cell nucleus was stained with DAPI (Invitrogen). Images were captured using Leica fluorescence optical microscope.
5
Immunofluorescent Assay for γH2AX Detection
6
Immunofluorescent Assay for γH2AX Detection
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Cells were washed with cold PBS and fixed with 4% PMSF (Sangon) for 15 min at room temperature. Then, cells were treated with 0.3% Triton X-100 for cell perforation at room temperature for 15 min, and were incubated with blocking buffer (3% BSA in PBS) for 1 hour, followed by incubation at 4°C overnight with the primary antibody against γH2AX, and Alex Fluor 594 (Green) conjugated secondary antibody (Invitrogen) at room temperature for 1 hour. Cell nucleus was stained with DAPI (Invitrogen). Images were captured using Leica fluorescence optical microscope.
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