Immunofluorescence analysis was performed as described by us earlier [31 (link), 34 (link)]. U87MG cells were plated in a 24-well plate on a coverslip at a density of 0.1 × 106cells/well and allowed to attach overnight followed by treatment with 2.5 μM and 5 μM penfluridol for additional 48 h. The cells were fixed with formalin and Triton-X100 solutions to permeabilized the cells. After blocking with 6% goat serum in 1%BSA, cells were incubated overnight with antibody against OCT4 (1:400). Cells were washed with PBS and again incubated overnight with Actin antibody (1:2000). Next day cells were incubated with AlexaFluor 488 secondary antibody (Invitrogen, Carlsbad, CA) as well as AlexaFlour 594 secondary antibody (Invitrogen, Carlsbad, CA) separately, after washing three times with PBS in between and after incubation. Coverslips were mounted on slides (mounting media with DAPI) and images were taken using multi photon confocal microscope (Nikon).
Multi photon confocal microscope
Manufactured by Nikon
The Nikon Multi Photon Confocal Microscope is a versatile scientific instrument that utilizes multi-photon excitation and confocal imaging technology. The core function of this microscope is to enable high-resolution, three-dimensional imaging of biological samples by selectively exciting fluorophores within the specimen.
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3 protocols using multi photon confocal microscope
1
Immunofluorescence Analysis of OCT4 and Actin
2
Immunofluorescence Assay of pAkt in AsPC1 Cells
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AsPC1 cells were plated on a coverslip in a 24-well plate at a concentration of 4 × 104 cells per well and allowed to attach overnight. Cells were then treated with 2.5 μM PVT for 48 h. For the immunofluorescence analysis, cells were washed with 1× PBS twice and then fixed with 4% paraformaldehyde (PFA) for 15 min. PFA-fixed cells were washed 3 times with 1× PBS and permeabilized using 0.1% Triton-X 100 for 5 min. Cells were washed with 1× PBS and then blocked using 6% goat serum in 1% BSA and 0.1% Triton-X 100 for 1 h. Cells were then incubated with antibody against anti-pAkt(Ser473) (1:400) overnight. The next day, cells were washed twice with 1× PBS and incubated with anti-rabbit Alexa Fluor 488 conjugate secondary antibody (1:1,000) in 3% goat serum prepared in 0.5% BSA for 1 h. Cells were washed and incubated with Alexa Fluor 594 phalloidin (1:500) in 3% BSA for 15 min. Cells were washed and coverslips were inversely mounted on the slides using DAPI mounting medium. Images were taken in 512 pixels using a multiphoton confocal microscope (Nikon).
3
Quantifying Cellular Internalization of DSF
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The cellular internalization was quantified through an established HPLC method. Briefly, DAOY and T98G cells were incubated in a 6-well plate until they grew to 70% confluency. Next, they were treated with 40 μM of DSF or DSFNPs for 0.5, 1, and 2 h or with 10, 20, and 40 μM of DSF or DSFNPs for 1 h. Cells were washed with cold PBS and kept at −80°C for cell lysis. DSF and cellular proteins were extracted by sonication followed by centrifugation. The DSF present in the supernatant was extracted into methanol followed by HPLC for quantitation. The drug amount was normalized to the protein content in each sample. Using a NIR dye encapsulated nanoparticles we performed confocal imaging as well as flow cytometric analysis to understand the extent of uptake in a time-dependent manner. Cells were incubated with HITCNPs, a NIR dye encapsulated NPs for 30 min, 1 h and 2 h followed by washing and fixing with 4% formaldehyde. Next, the cells were analyzed by flow cytometry or counterstained with DAPI followed by imaging under a multi-photon confocal microscope (Nikon Instruments Inc).
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