Alexa fluor 594 conjugated anti mouse antibody

To inhibit V-type ATPase transport to the membrane (Matsumoto and Nakanishi-Matsui, 2019 (link)), osteoclasts on the fifth day of differentiation were incubated with 100 nM bafilomycin A1 (19-148; Sigma-Aldrich) for 3 hr. Then, immunofluorescence staining was performed to visualize the localization of the V-type ATPase in bafilomycin A1-treated and untreated cells. The cells were fixed using a 4% PFA solution (PC2031-100; Biosesang, Gyeonggi-do, Korea) and permeabilized using 0.05% Triton X-100 at room temperature for 5 min. The cells were incubated with anti-V-type ATPase antibody (SAB1402125-100UG; Sigma-Aldrich) at room temperature for 1 hr, and then stained with the Alexa Fluor 594-conjugated anti-mouse antibody (A-21044; Invitrogen) at room temperature for 30 min. Finally, cells were mounted using Antifade Mountant with DAPI (P36962; Invitrogen). Fluorescence images were observed under a ZEISS confocal microscope (LSM5; Carl Zeiss).

Male mice were deeply anesthetized followed by serial intracardial perfusions with 0.9% saline (100 mL/100 g), and 4% polyformaldehyde in phosphate buffer (100 mL/100 g). Whole brains were rapidly dissected and postfixed with 4% polyformaldehyde at 4°C overnight and cryoprotected with 30% sucrose at 4°C. Then, the brain tissues were embedded, frozen in OCT at −20°C, and sectioned into 30‐μm slices using a Leica microtome. Brain sections were permeabilized with 0.1% Triton X‐100 in PBS, blocked with 5% normal bovine serum in PBS, and incubated with primary antibodies anti‐CaMKII‐ɑ (1:1000, Invitrogen, USA), anti‐c‐Fos (1:1000, Abcam, USA) or anti‐SK2 (1:100, Alomone labs, Israel) at 4°C overnight. Subsequently, the brain slices were rinsed three times, incubated with an Alexa Fluor 488‐conjugated anti‐rabbit (1:1000, Invitrogen, USA) or Alexa Fluor 594‐conjugated anti‐mouse antibody (1:1000, Invitrogen, USA) at r/t for 2 h, and mounted on positively charged slides. Images were obtained with a confocal laser scanning microscope.

Cells were fixed for immunofluorescence staining, which was performed as described previously (16 (link)). Primary antibodies targeting EI24 (NBP2-13949, Novus, [Littleton, CO, USA]) or LC3 (#2775, Cell Signaling) and Alexa Fluor® 488-conjugated anti-rabbit antibodies (Molecular Probes, Carlsbad, CA, USA) were used. Double immunofluorescence staining was performed sequentially. First, the fixed cells were incubated with 1% bovine serum albumin and 0.02% Triton X-100 in phosphate-buffered saline (PBST–BSA) and anti-LC3 antibody (1:500, #2775, Cell Signaling) overnight at 4°C, followed by Alexa Fluor® 488-conjugated anti-rabbit antibodies (1:500, Molecular Probes) for 1 h at room temperature. Alternatively, the fixed cells were incubated with PBST-BSA and anti-LAMP1 antibody (1:500, ab25630, Abcam [Cambridge, UK]) for 3 h at room temperature, followed by Alexa Fluor® 594-conjugated anti-mouse antibodies (1:500, Molecular Probes) and Hoechst 33342 (10 μg/ml, Molecular Probes) for 10 min. The cells were washed three times with PBST for 10 min after each incubation step. After mounting, the cells were visualized under a confocal microscope (Carl Zeiss Microscopy GmbH, Jena, Germany). Pearson's co-localization coefficients were analyzed by ZEN 2.6 (blue edition) as described (17 (link)).