Confocal imaging system

Cells were cultured on a glass coverslip in 12 well plates and treated with compounds (for 24 h). After fixing the cells with pre-chilled absolute methanol at −20 °C for 15 min, cells were stained for 5 min with 25 nM DAPI (Invitrogen, Waltham, MA, USA), followed by two times PBS wash. The coverslips were then picked carefully and mounted on a glass slide, and observed under a confocal imaging system from Leica.

We used free-floating 20 μm brain sections for immunofluorescence staining. The brain sections were washed three times with PBS and blocked with 1% bovine serum albumin (w/v) and 0.3% Triton X-100 (v/v) for 30 min, and were incubated with primary antibodies at 4°C overnight. On the second day, after washed three times with PBS, the brain sections were incubated at room temperature with a mixture of labeled secondary antibodies for 2 h. Finally, DAPI (1:1,000; Sigma-Aldrich) was used for staining nuclei for 5 min followed by washed three times with PBS. Images were acquired with a Leica Confocal Imaging System.

Free-floating 20 μm brain sections were used for immunofluorescence staining. The brain sections were washed three times with PBS, blocked with 3% bovine serum albumin (BSA, w/v) and 0.3% Triton X-100 (v/v) for 1 h at room temperature, and then incubated with primary antibodies at 4°C overnight (anti-Tau N368, 1:500). On the second day, after washed with PBS three times, the slices were incubated at 37°C with a mixture of labeled secondary antibodies (Alexa Fluor® 488) for 1 h. After washing with PBS three times, DAPI (1:1000) was added for nucleus staining for 15 min. Images were captured using a Leica Confocal Imaging System.

Cells were cultured on glass coverslips (MGF-slides, Germany) for 24 h and then incubated with appropriate Bortezomib concentrations. After this interval, they were gently rinsed with PBS and fixed in a 4% paraformaldehyde aqueous solution for 15 min at room temperature. Subsequently, they were rinsed three times with PBS, permeabilized for 15 min in a PBS solution containing 0.1% Triton X-100 and then blocked in a PBS solution containing 5% skimmed milk for 1 h at room temperature. Cells were then incubated for 1 h, at room temperature with the primary antibodies/fluorophores (Table 2).
After rinsing with PBS Tween-20, the coverslips were incubated with secondary antibodies; Donkey anti-Rabbit IgG (H+L) Highly Cross-Adsorbed antibody, Alexa Fluor™ 647 (Invitrogen, #A-31573) or Goat anti-Mouse IgG (H+L) Cross-Adsorbed secondary antibody, Alexa Fluor™ 488 (Invitrogen, #A-11001) (Dilution 1:500) in permeabilization buffer. After rinsing three times in PBS, cells were mounted using Mowiol 4–88® (Sigma). To detect autophagy, live cells grown on coverslips were incubated with Lysotracker RED (Invitrogen) for 15 min and then immediately imaged. Labeling was performed using Leica Confocal Imaging System and photographs were taken using the LAS X software. Lysotracker staining of live cells was also used to quantify the acidic protein content using flow cytometry.

HT22 cells were cultured in sterile 24-well culture dishes containing 14 mm cover-glass. Cells were treated with PRO-Br (12.5 and 25 μM), and DMSO (0.1%), for 24 h. Cells were fixed with paraformaldehyde (4%), permeabilized with triton-X-100 (0.1%), and blocked with BSA (2%). LAMP2A antibody (1:500), and HSC70 antibody (1:500) was added to the processed cells and incubated overnight at four°C. Alexa Fluor^® 488 (green) and Alexa Fluor^® 594 (red) secondary antibodies (1:400) were added and incubated for 1.5 h at room temperature. FluorSave reagent (Merck Millipore, United States) was added, then cover-slipped, and images were captured by a Leica confocal imaging system.