For immunofluorescence (IF) staining of OPCs, cells were permeabilized with 0.2% triton-X100 for 5 min followed by blocking with 5% normal donkey serum for 1 hour at room temperature. Rabbit anti-neural/glial antigen 2 (NG2, 1:300, ab5320) and rat anti-Bromodeoxyuridine/5-bromo-2’-deoxyuridine (BrdU, 1:200, ab6326) monoclonal antibody staining was performed overnight at 4°C followed by rinsing with PBS and incubating with goat anti-rabbit (1:200, AF488, Invitrogen, A11008) and goat anti-rat (1:250, Cy3, Jackson Immunoresearch, 112–165-167) secondary antibodies at room temperature for 1 hour. Slides were coverslipped using mounting media with DAPI (4′,6-diamidino-2-phenylindole) counterstain (Vector Hardset Antifade Mounting Media), and stored at −20°C until imaging. Slides were scanned into digital files with a Zeiss Axio Scan.Z1 slide scanner at 20x magnification.
Hardset antifade mounting media
Manufactured by Vector Laboratories
Hardset Antifade Mounting Media is a laboratory product designed to protect fluorescent signals and preserve sample integrity. It is formulated to harden after application, creating a stable mounting solution for microscopy and imaging applications.
Automatically generated - may contain errors
Lab products found in correlation
3 protocols using hardset antifade mounting media
1
Immunofluorescence Staining of Oligodendrocyte Progenitor Cells
2
Evaluating Macrophage Heterogeneity via Immunofluorescence
3
Immunofluorescence Analysis of HCT116 Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
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HCT116 cells were seeded onto sterile
18 mm coverslips in 12 well plates and incubated for 24 h. Cells were
then treated with DMSO or 1 nM OSW-1 compound for the indicated times.
Cells were fixed using 4% paraformaldehyde and permeabilized using
0.5% Triton X-100. Image-iT FX signal enhancer (Thermo I36933) was
added to the coverslips, followed by incubation with 1% BSA for blocking.
Primary antibody was then added, and the coverslips were incubated
overnight at 4 °C. Secondary antibody was incubated in darkness
at RT for 1 h. After washing, the coverslip was soaked in 300 nM DAPI
(Thermo D1306) solution for 10 min, and coverslips were mounted onto
glass slides using VECTASHEILD HardSet Antifade mounting media (VECTOR
laboratories H-1400). Imaging was performed with a Lecia SP8 using
a 63× glycerol objective with 2× digital zoom. Images were
analyzed with ImageJ software.
18 mm coverslips in 12 well plates and incubated for 24 h. Cells were
then treated with DMSO or 1 nM OSW-1 compound for the indicated times.
Cells were fixed using 4% paraformaldehyde and permeabilized using
0.5% Triton X-100. Image-iT FX signal enhancer (Thermo I36933) was
added to the coverslips, followed by incubation with 1% BSA for blocking.
Primary antibody was then added, and the coverslips were incubated
overnight at 4 °C. Secondary antibody was incubated in darkness
at RT for 1 h. After washing, the coverslip was soaked in 300 nM DAPI
(Thermo D1306) solution for 10 min, and coverslips were mounted onto
glass slides using VECTASHEILD HardSet Antifade mounting media (VECTOR
laboratories H-1400). Imaging was performed with a Lecia SP8 using
a 63× glycerol objective with 2× digital zoom. Images were
analyzed with ImageJ software.
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