Paraformaldehyde pbs

CHO-GolEGF-tmco cells were grown on the surface of collagen-coated round-type coverslip (Matsunami, CO13001) in DMEM with 10% FBS including G418 (100 μg/ml), Zeocin (300 μg/ml) and Blasticidin (10 μg/ml). After 60% confluent, tetracycline was added (10 μg/ml) and another 24 h culture was performed to induce the expression of TMCO5. In the control experiment, the addition of tetracycline was omitted not to induce the expression of TMCO5. After 24 h culture, cells attached to a coverslip were washed with PBS (10x D-PBS (-), Fujifilm, 048–29805) and then fixed with 4% paraformaldehyde-PBS (Fujifilm, 161–20141) for 10 min at room temperature. After being washed with TBST, they were blocked with the Blocking solution, then reacted with culture supernatant of RTm01 antibody mixed with 1:2,000-diluted mouse anti-β-tubulin antibody (abcam, ab131205) for 1 h. After washing twice with TBST, Alexa 647-labeled anti-rat IgG (CST Japan) and Alexa 594-labeled anti-mouse IgG (CST Japan), each diluted 2,000 times with blocking solution, were reacted for 1 h. After 3 times washing with TBST for 5 min, DAPI (4′, 6-diamidino-2-phenylindole) (ThermoFisher, D1306) was used for fluorescent nuclear staining in accordance with the instruction manual. The samples were observed with a confocal microscope (TCS SP8, Leica).

Confocal microscopy was used to observe the localization of influenza H1N1 virus and to visually determine the antiviral effects of Cu MPs and Cu NPs in MDCK or A549 cells. After being incubated with Cu MPs or Cu NPs for the indicated times, viruses were used to infect cells for 1 h. Then, cells were washed with PBS and fixed in paraformaldehyde PBS (Fujifilm, Osaka, Japan) for 20 min at RT. Cells were then permeabilized with 0.1% Triton X-100 in PBS for 10 min and blocked with 1% bovine serum albumin (BSA) in PBS-T for 30 min at RT. After blocking, cells were incubated with mouse monoclonal antibody against viral NuP (1:200) (Abcam, Cambridge, UK) overnight at 4 °C and goat antibody mouse (1:1000) (Thermo Fisher Scientific, Rockford, IL, USA) for 1 h at RT. Nuclei were stained using 4′,6-diamidino-2-phenylindole (DAPI). Fluorescence was visualized with a Carl Zeiss LSM5 EXCITER fluorescence microscope (Carl Zeiss, Oberkochen, Germany).

TCID₅₀ was used to measure the titration and infectivity of viruses. Briefly, cells were seeded into 96-well plates and incubated for 24 h. Viruses were incubated with the absence or presence of Cu MPs and Cu NPs for 10 min, and 100 µL of incubated samples was used to infect cells as described in Section 2.6. After infection, cells were incubated for additional 48 h. After 48 h, cells were washed with phosphate buffered solution (PBS), fixed with 4% paraformaldehyde-PBS (Fujifilm, Osaka, Japan) for 20 min, and stained with crystal violet solution for 10 min at room temperature (RT). The TCID₅₀ per mL was calculated according to Reed and Muench method [26 (link)].

RPMI culture medium containing 0.3% agarose (Lonza, Basel, Switzerland) with LM05-shGFP or LM05-shNIK cells (4.0 × 10⁴ cells/well) over a bottom layer of 0.6% agarose in RPMI culture medium were plated in each well of a 6-well plate and cultured for 3 weeks. Colonies were fixed with 4% paraformaldehyde-PBS (Fujifilm Wako Pure Chemical Corporation) for 1 h and stained with 0.005% crystal violet solution (Fujifilm Wako Pure Chemical Corporation) for 30 min. After removing the overdyed region with Milli-Q water, the colony images were acquired with a digital camera (Nikon Corporation, Tokyo, Japan), and colony numbers were calculated with ImageJ software (National Institutes of Health, MD, USA).

The cells were fixed with 4% paraformaldehyde/PBS (Wako) and permeated through the membrane with 0.5% Triton X-100/tris-buffered saline (TBS) for 1 minutes. The cells were blocked with 1% bovine serum albumin (BSA) and 10% goat serum/TBS for 1 hour at 4 °C. Then, it was made to react with the following antibodies: γH2AX (1: 1,000, 05-636, Millipore, Temecula, CA, USA), phospho-(Ser/Thr) ATM/ATR substrate (1: 500, 2851, Cell Signaling Technology, Danver MA, USA), 53BP1 (1: 250, sc-58749, Santa Cruz). The secondary antibody was reacted using Alexa488 and Alexa594 (Thermo Fisher Scientific), and the nucleus was stained using DAPI (Dojindo, Tokyo, Japan). After immunostaining, DNA damage-positive cells were quantified by observation using a fluorescence microscope (Carl Zeiss, Oberkochen, Germany).

The cells were fixed on days 3, 7, 14, 21, and 28 after incubation with an osteogenic differentiation medium, and ALP staining was performed using the following methods. After fixation with 4% paraformaldehyde PBS (Fujifilm Wako Pure Chemical, Osaka, Japan) for 10 min, the cells were incubated with PBS containing 0.05% Tween-20 (Roche Diagnostics) (washing buffer). After removing the washing buffer, the cells were incubated with ALP staining solution (Fujifilm Wako Pure Chemical Industries, Ltd.) at room temperature under light-resistant conditions for 10 min.
For the quantitative testing of ALP, the cells were harvested on days 3, 7, 14, 21, and 28 using the pNPP Phosphatase Assay Kit (AnaSpec, Fremont, CA, USA) after incubation with an osteogenic differentiation induction medium. The harvested cells were homogenized using a Sonic Vibra Cell (Sonic & Materials, Newtown, CT, USA), and the supernatant was collected and used as the sample. The sample was mixed with 50 μL of the pNPP substrate solution in a 96-well plate (CORNING), and the absorbance was determined at a wavelength of 405 nm using a microplate reader MultiskanTM FC (Thermo Fisher Scientific).

After the cells were seeded onto 8-well chamber slides, testosterone and metformin were added. The cells were washed with PBS (−) and fixed in 4% paraformaldehyde/PBS (#163–20,145, Wako) at room temperature for 15 min and permeabilized with PBS/0.5% Triton-X. The primary antibody was diluted with 3% bovine serum albumin (BSA)/PBS (−) and incubated at 4 °C overnight. The secondary antibody (donkey anti-rabbit IgG H&L [Alexa Fluor 488] donkey polyclonal secondary antibody, #ab150073; Abcam) was incubated for 60 min at room temperature. After probing with each antibody, the slides were finally encapsulated with 4′ 6-diamidino-2-phenylindole (DAPI)-added mounting agent, Pro Long Gold Antifade Mountant with DAPI (#P36941, Thermo Fisher Scientific). A confocal laser microscope system (OLYMPUS FV1000, Olympus) was used for imaging.