Cells grown over Matrigel‐coated coverslips were fixed with 4% paraformaldehyde (SantaCruz Biotechnology #SC‐281692), permeabilized with 0.1% Triton X‐100 (Sigma‐Aldrich #T8787), blocked in PBS (ThermoFisher Scientific #A12856‐01) with 1% bovine serum albumin (BSA, Sigma‐Aldrich #A8806) for 30 minutes at 37°C and incubated with the primary antibody overnight at 4°C. Cells were subsequently incubated with the secondary antibody for 30 minutes at 37°C and mounted with ProLong Gold Antifade Mountant with 4′,6‐diamidino‐2‐phenylindole (DAPI, ThermoFisher Scientific #P36930). Primary and secondary antibodies are listed in Table S1 . For Ki‐67 detection, we first performed an antigen retrieval step in which cells were heated for 10 seconds in a microwave with citrate buffer pH 6.0 (Sigma‐Aldrich #C9999) letting cells cool down 20 minutes. Acquisition of fluorescence images was performed in a Leica TCS‐SP5 or a Nikon Eclipse Ti fluorescence microscope. Images were processed using the Adobe Photoshop CS5 or ImageJ software.28 Positive cells were counted using the ImageJ software from at least three random fields per preparation.
T8787
Manufactured by Thermo Fisher Scientific
The T8787 is a laboratory equipment product manufactured by Thermo Fisher Scientific. It is designed for precise temperature regulation and control in various scientific applications. The core function of the T8787 is to maintain a stable and accurate temperature environment for experimental procedures or sample processing.
Automatically generated - may contain errors
2 protocols using t8787
1
Immunofluorescence Imaging of Cells
2
Immunoprecipitation of MYC Protein
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Total cell lysate was harvested by scraping cells in 1 ml of IP lysis buffer (50 mM HEPES, pH 7.9 [Sigma-Aldrich, PHG0001], 250 mM NaCl [Sigma-Aldrich, S3014], 5 mM Ethylenediaminetetraacetic acid [EDTA; Sigma-Aldrich, E6758], 1% NP-40 [Abcam, ab142227], 1% Triton X-100 [Sigma-Aldrich, T8787]) containing complete Pierce Proteinase Inhibitor (Thermo Scientific, A32963). Samples were collected and placed on ice for 30 min before centrifugation at 20,000 × g for 12 min. 1 ml of supernatant from each sample was incubated with anti-MYC antibody for 4 h. After incubation with the antibody, protein A agarose beads (Sigma-Aldrich, PROTAA-RO) were added into the lysate and incubated at 4°C overnight. The beads were collected by centrifuging at 1,500 × g for 1 minute and washed with 1 ml IP lysis buffer for 6 times and eluted with 2X sample buffer.
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