P24-25 WT, Kcnb1R/+ and Kcnb1R/R mice were used for immunohistochemistry, while Kcnb1fs mice were not included in this analysis because epitopes for the immunolabeling-suitable Kv2.1 antibodies are all downstream of G379VfsTer6 frameshift. Animals were deeply anesthetized with pentobarbital and transcardially perfused with 4% formaldehyde prepared from paraformaldehyde, in 0.1 M sodium phosphate buffer pH 7.4 (0.1 M PB). Sagittal brain sections (30μm thick) were prepared and immunolabeled using free-floating methods as previously described (Rhodes et al., 2004 (link); Speca et al., 2014 (link); Palacio et al., 2017 ). Briefly, free floating sections were blocked and permeabilized. Sections were incubated with primary antibodies (see Table 2 for antibody details) and then immunolabeled with Alexa-conjugated fluorescent IgG subclass-specific secondary antibodies and Hoechst 33258 DNA stain at 200 ng/mL (ThermoFisher Cat# H21491). Images were taken using the same exposure time to compare the signal intensity directly. Images were identically processed in Photoshop to maintain consistency between samples. For further details, see supplemental materials and methods .
H21491
Manufactured by Thermo Fisher Scientific
The H21491 is a laboratory centrifuge designed for general-purpose applications. It is a compact and reliable instrument that provides consistent and accurate separation of samples. The centrifuge is capable of accommodating a range of sample volumes and can achieve high-speed rotation to facilitate efficient separation.
Automatically generated - may contain errors
4 protocols using h21491
1
Kcnb1 Mutant Mouse Immunohistochemistry
2
Immunofluorescence Staining of Cells
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Cells were seeded on glass coverslips in 24-well plates (Costar) at a density of 25,000 cells per well. After the incubation time, the medium was removed and cells were then fixed during 10 min with 4% paraformaldehyde (PFA) in phosphate buffer saline (PBS) before being washed three times with PBS and permeabilized with PBS-triton 1% for 5 min at room temperature. After permeabilization, cells were washed three times for 10 min with PBS-BSA 2% (Bovine Serum Albumin) (Santa Cruz Biotechnology) then incubated with the primary antibody overnight at 4 °C in the dark (Table 2 ). The next day, cells were washed three times with PBS-BSA 2% before being incubated for 1 h with the secondary antibody and Hoechst (Thermo Fisher Scientific H-21491) at 2 μg/mL at room temperature in the dark. Thereafter, cells were washed three times with PBS-BSA 2% and once with PBS. The coverslips were finally mounted on slides in Mowiol mounting solution (Sigma) warmed at 57 °C. Slides were observed by confocal microscopy (SP5, Leica).
3
Kcnb1 Mutant Mouse Immunohistochemistry
4
Phospho-histone H2AX Immunofluorescence Assay
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Cells were seeded on glass coverslips in 24-well plates (Costar) at a density of 40,000 cells per well. 24 h later, the medium was removed and replaced for the different sequences of incubation with afatinib and/or cisplatin. After 48 h of incubation, the medium was removed and cells were fixed, permeabilized, and labeled following the procedure described previously (48 (link)). The primary antibody used was rabbit anti-phospho-histone H2AX (9664, cell signaling, Leiden, Netherlands), diluted at 1:400 in bovine serum albumin (BSA) 2% with phosphate buffer saline (PBS) and incubated overnight at 4°C in dark. The secondary antibody used was Alexia 488 nm anti-rabbit diluted at 1:1000 (Fisher Scientific). Nuclei were stained with Hoechst (Thermo Fisher Scientific H-21491) at 2 μg/mL at room temperature in the dark for 1 h. The coverslips were finally mounted on slides in Mowiol mounting solution (Sigma) warmed at 57°C. Slides were kept at 4°C to be observed later under a confocal laser scanning fluorescence microscope (SP5, Leica) with a constant photomultiplier.
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