Tcs spe 2 confocal microscope

To evaluate the progression of amyloid pathology in this model, a different set of mice (age 4, 8 and 12 months) were euthanized via CO₂ inhalation and transcardially perfused with ice-cold PBS. Brains were removed, and hemispheres were separated along the midline. Each hemisphere was then drop-fixed in 4% PFA for 48 h, cryoprotected in 30% sucrose +0.05% sodium azide, and sectioned at 40 μm on a Leica SM2000R freezing microtome. Amylo-Glo (TR-300-AG; Biosensis) staining was performed according to the manufacturer’s instructions and as before (Spangenberg et al., 2019 (link)). Immunostained sections were mounted and coverslipped, then images were obtained using a Leica TCS SPE-II confocal microscope and LAS-X software. One 20× field-of-view (FOV) of the hippocampal CA1 was captured per mouse. Plaque number, area and intensity were determined using the surfaces module in Imaris v9.2.

For immunocytochemistry of active integrin‐β1, cells were seeded on sterile coverslips in a six‐well plate at a density of 173,000 cells per well. The coverslips were fixed in 4% paraformaldehyde and permeabilized in 0.1% Triton X‐100 on ice for 1 min. After blocking in 5% BSA‐1X PBS, active integrin‐β1 antibody (1:100) was added for 1 h followed by 1h incubation with secondary antibody. After repeated washing in 1X PBS, cells were stained with the DNA stain DAPI (4,6‐diamidino‐2‐phenylindole dihydrochloride; #1023627001; Roche) and mounted using Prolong gold anti‐fade mount media (#P36930; Invitrogen) on glass slides. Imaging and z‐stacks (1‐µm z‐slice) were acquired using a Leica TCS SPEII confocal microscope maintaining consistent acquisition parameters between experiments.

Keratinocytes were cultured on glass coverslips, treated with etoposide (10 μm) for 8 h and fixed in 4% PFA for 10 min at room temperature. For 5-bromo-2′-deoxyuridine (BrdU) detection, cells were treated with BrdU (Sigma, St Louis, MO, USA, B5002) 6 h before fixation. Cells were incubated with 2 M HCl for 20 min, followed by addition of 0.1 M sodium borate (Na₂B₄O₇), pH 8.5 for 2 min. Cells were washed with PBS and permeabilized with 0.2% triton/PBS for 5 min. Immunofluorescence staining was done by anti-BrdU-FITC for 2 h. Coverslips were mounted on slides using Fluoroshield mounting medium containing DAPI (Sigma, F6057).
Cells were fixed with 4% PFA/0.1% triton/PBS for 10 min at room temperature for immunofluorescence staining for γ-H2Ax and pH3. Cells were blocked with 4% BSA/TBS for 30 min, and incubated for 2 h with primary antibodies dissolved in 2% BSA/TBS. Cells were washed and incubated with a secondary antibody conjugated with Alexa-Fluor 488 for 1 h. DAPI was used as nuclear counterstaining. Images were taken using a Leica TCS SPE-II confocal microscope and analyzed using Fiji software.^{46 (link)} Used antibodies are in Supplementary Table S2.