Can get signal solution 2

After perfusion–fixation of mice under isoflurane inhalation, brains were extracted, and 20 µm thick frozen sections were prepared using a cryostat (Leica Biosystems, Wetzlar, Germany). The sections were permeabilized by washing three times for 5 min in phosphate-buffered saline (PBS) containing 0.3% TritonX-100 (PBST) and then blocked for 2 h at room temperature in PBST containing 1% BSA. The brain sections were then incubated overnight at 4 °C with rabbit anti-ionized calcium-binding adapter molecule-1 (Iba1) (Fujifilm Wako, Osaka, Japan) and mouse anti-glial fibrillary acidic protein (GFAP)-Cy3 (Sigma-Aldrich), diluted 1000- and 400-fold, respectively, in PBST containing 1% BSA. After washing with PBST three times, the sections were incubated in Can Get Signal Solution II (Toyobo, Osaka, Japan) containing Amylo-Glo (Biosensis, Thebarton, Australia), a fluorescent probe for Aβ plaque, and 500-fold-diluted goat-anti-rabbit-Cy5 (AnaSpec, Fremont, CA, USA) at room temperature for 2 h. Thereafter, the brain sections were washed three times for 5 min with PBST and sealed in VECTASHIELD Mounting Medium (Vector Laboratories, Newark, CA, USA). An all-in-one fluorescence microscope (BZ-X800; Keyence, Osaka, Japan) with BZ-X800 filter DAPI for Amylo-Glo, BZ-X800 filter green fluorescent protein for GFAP, and BZ-X800 filter Cy5 for Iba1 was used to detect signals.

Western blots were performed with whole‐cell lysates. Antibodies against tubulin and HLA‐DR (Abcam PLC, Cambridge, UK) were used with a working dilution in Can Get Signal solution I (Toyobo, Tokyo, Japan), and secondary antibodies (anti‐rabbit IgG horseradish peroxidase from Cell Signaling, Danvers, MA, USA) were used at 1 : 2000 dilutions in Can Get Signal solution II (Toyobo). Signals were observed via ECL Western blotting Detection Reagents (GE Healthcare UK Ltd., Buckinghamshire, UK). The western blot images were captured using the Amersham Imager 600 (GE Healthcare UK Ltd.). Quantification (%) was calculated according to the following formula ‘[(band intensity of HLA‐DR)/ (band intensity of Tubulin bands)] × 100’ (%) by using imagej software (NIH, Bethesda, MD, USA).

Western blotting was conducted according to our previously described methods (Takanami et al., 2016 (link)). Membranes were blocked with the PVDF blocking reagent from the Can Get Signal kit (Toyobo) for 30 min at room temperature and then incubated overnight at 4°C in Can Get Signal Solution 1 (Toyobo) containing a 1:1,000 dilution of rabbit polyclonal antibody against human GRPR (GTX100015, GeneTex). Blotted membranes were washed three times with 0.05% Tween 20 in Tris-HCI buffered saline (TBST) and incubated with HRP-conjugated goat polyclonal antibody against rabbit IgG (Bio-Rad Laboratories) at a 1:10,000 dilution in Can Get Signal Solution 2 (Toyobo) for 1 h at room temperature. After washing five times with TBST, blots were visualized by the Immun-Star WesternC Chemiluminescence Kit (Bio-Rad Laboratories). Images of blots were detected by ChemiDoc XRS+ System with Image Lab Software (Bio-Rad Laboratories) and adjusted slightly for brightness and contrast to provide a uniform background.

On feeding day 58, the mouse brain cortex was homogenized with M-PER (Thermo Fisher Scientific, Waltham, MA, USA) containing 1 × Halt protease and phosphatase inhibitor cocktail (Thermo Fisher Scientific). Samples were mixed with NuPAGE lithium dodecyl sulfate sample buffer (Thermo Fisher Scientific) containing 5% 2-mercaptoethanol (Wako, Osaka, Japan) at 75 °C for 5 min and loaded onto a 14% sodium dodecyl sulfate polyacrylamide gel (SDS–PAGE) (50 µg/lane). After electrophoresis, proteins in the gel were transferred to a nitrocellulose membrane (Bio-Rad, Hercules, CA, USA). After blocking with 0.1% T-TBS containing 5% skim milk (Wako) at room temperature, the membrane was incubated with a mouse monoclonal anti-Aβ antibody (1:1000, clone 6E10, BioLegend, San Diego, CA, USA) or mouse monoclonal anti-GAPDH (1:10000, clone 5A12, Wako) in Can Get Signal solution 1 (Toyobo, Osaka, Japan) overnight at 4 °C. After washing with 0.1% T-TBS, the membrane was incubated with a horseradish peroxidase-conjugated secondary antibody against mouse IgG (1:2000; Cat. No. sc-2005, Santa Cruz) in Can Get Signal Solution 2 (Toyobo) for 2 h at room temperature. After washing, chemiluminescence on the membrane was detected by ECL Prime Western Blotting Detection Reagent (GE Healthcare, Chicago, IL, USA) using an ImageQuant LAS 4000 system (GE Healthcare).

Cell lysates of all five cell lines were prepared in lysis buffer containing 50 mM Tris-HCL, 150 mM NaCl, 5 mM EDTA, 0.5% TritonX-100, 0.05% sodium dodecyl sulfate (SDS), 0.5% sodium deoxycholate, 2 mM phenylmethylsulfonyl fluoride and 1 mM Na₃VO₄. The lysates were separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting analysis was performed as previously described [20 (link)].
Membranes were incubated with a 1:1000 dilution of primary antibodies in Can Get Signal solution 1 (TOYOBO, Osaka, Japan), followed by corresponding secondary antibodies (1:1000 dilution with Can Get Signal solution 2). Primary antibodies were obtained from Cell Signaling Technology (cleaved-PARP (cPARP), p21, acetyl-H4 (Lys12), E-cadherin; Danvers, MA, USA), Sigma-Aldrich (α-tubulin, acetyl-α-tubulin (Lys40); St. Louis, Mo, USA), Horse-radish peroxidase-conjugated mouse and rabbit antibodies were from GE Healthcare Life Sciences (Piscataway, NJ).