Prolong gold antifade mounting medium with dapi

For immunostaining, HEK293T cells were cultured and transfected in eight-chamber slides purchased from BD Bioscience (San Jose, CA). Cells were fixed with 4% formaldehyde for 15 min at room temperature and blocked with 5% fetal bovine serum and 0.2% Triton-X 100 in 1X PBS for 1 h at room temperature. Staining was carried out by incubating overnight with primary antibodies (1: 500) in 1% BSA and 0.2% Triton-X 100 in 1X PBS. The slides were rinsed three times with 1X PBS and incubated with fluorochrome-conjugated secondary antibodies (1:1,000) in 1% BSA and 0.2% Triton-X 100 for 1 h at room temperature. The slides were rinsed three times with 1X PBS, air-dried. The cells were coated with ProLong Gold antifade mounting medium with DAPI (4',6-diamidino-2-phenylindole, 100 ng/ml) (Life Technologies, P-36931), overlaid with coverslips and the mounting medium was allowed to cure at least overnight before imaging. Images were acquired on an LSM 710 laser-scanning confocal microscope.

HEL, U937, and NB4 cells were grown on cover slips coated with poly-L-lysine (1mg/mL), fixed with 4% paraformaldehyde, permeabilized with 0.5% Triton X-PBS and blocked with 3% bovine serum albumin (BSA) PBS. Cells were then incubated overnight at 4°C with anti-IRS2 or anti-JAK2 antibodies (1:200 in 1% BSA PBS), followed by incubation with secondary antibody (1:400 in 1% BSA PBS) for 2h at room temperature. All incubations were followed by three 5min PBS washes. The slides were mounted in ProLong Gold Anti-Fade Mounting Medium with DAPI (Life Technologies, Carlsbad, USA). Images were generated using a confocal laser-scanning microscope (LSM 510, Carl Zeiss, Welwyn Garden City, UK). The “colocalization finder” plug in of Image J quantification software (U.S. National Institutes of Health, Bethesda, USA) was used for the analysis of JAK2 and IRS2 colocalization analysis.

Cells were seeded at high density in 6-well plates containing glass coverslips. After 24 h, cells were treated with 1 µM DOX for 0 h (Control), 4 h, 8 h or 24 h. Then, cells were washed twice with phosphate-buffered saline (PBS; Corning, NY, USA) and fixed in 4% paraformaldehyde (PFA; Sigma-Aldrich, Saint-Louis, MO, USA) in PBS for 20 min at room temperature. Cells were then washed twice with PBS plus 0.1% Tween 20 (Sigma-Aldrich), permeabilized in PBS plus 0.1% Triton X-100 (Sigma-Aldrich) for 20 min and washed again with PBS. Slides were blocked in 10% BSA (VWR, Radnor, PA) for 30 min and incubated overnight at 4 °C with the primary antibody anti-γ-H2AX ([05–636], 1:500 dilution) from Merck KGaA (Darmstadt, Germany). Then, the slides were washed 3 times with PBS plus 0.1% Tween 20 and incubated with the secondary antibody Alexa Fluor 488 goat anti-mouse IgG ([A-11001], 1:1000 dilution) from Invitrogen (Waltham, MA, USA) for 1 h. Later, cells were washed another 4 times with PBS and, finally, coverslips were mounted in ProLong^® Gold Antifade Mounting medium with DAPI (Life Technologies, Carlsbad, CA, USA) and analyzed by fluorescence microscopy. The number of γ-H2AX foci per nuclei was counted in at least 100 cells of each condition using the ImageJ 2.1.0 software and the Cell Profiler 4.2.1 (Broad Institute, Cambridge, MA, USA) [53 (link)].

A549 cells were seeded into 24-well
plates (Costar, Cat# 3524) containing glass coverslips (Menzel Gläser,
Cat# MENZCB00130RAC) and incubated with 5 μM of final concentration
of probes JYQ-192, JYQ-196, and JYQ-197 for fluorescence confocal microscopy of fixed samples. Fixation
was performed in 3.7% formaldehyde (acid-free, Merck Millipore) in
phosphate-buffered saline (PBS) for 20 min. After washing 3×
with PBS, cells were mounted using a ProLong Gold antifade Mounting
medium with DAPI (Life Technologies, Cat# P36941). Samples were imaged
using a Leica SP8 microscope equipped with appropriate solid-state
lasers, HCX PL 63 times magnification oil emersion objectives and
HyD detectors. Data were collected using a digital zoom in 1 in 1024
by 1024 scanning format with line averaging. Postcollection image
processing was performed using the Fiji software.

Sections from WT Fisher and TgF344-AD rats were incubated overnight at 22 ± 2°C with the following primary antibodies, (1) 1:500 dilution of goat anti-PIEZO1 (N-15; Santa Cruz, sc-164319, RRID: AB_10842990), an affinity-purified goat polyclonal antibody raised against a peptide mapping to the N-terminus of PIEZO1 of human origin; (2) 1:1000 dilution of rabbit anti-amyloid β_1-42 (mOC98, Abcam, ab201061), a conformation-specific antibody that recognizes a discontinuous epitope of Aβ that maps to segments AEFRHD and EDVGSNK; (3) 1:1000 dilution of chicken anti-GFAP (Abcam, ab4674, RRID: AB_304558). Slides were then washed in PBS and incubated for 4 h at 22°C with the following secondary antibodies, (1) donkey anti-goat Alexa 488 (Abcam, ab150133), (2) donkey anti-rabbit 555 (Sigma, SAB4600061), (3) donkey anti-chicken IgY (H + L) CF^TM 633 (Sigma, SAB4600127). Finally, slides were washed and coverslipped using ProLong^® Gold antifade mounting medium with DAPI (Life Technologies, United Kingdom, P36935).