Antibody diluent solution

Immunostaining was performed on the brain slices of animal that were stimulated using the rodent coil. Rats were sacrificed and perfused with 4% PFA. Thirty-micron sections that were near bregma 0 were selected and washed three times, for five minutes each in PBS. Blocking was performed using 10% normal goat serum (Sigma, Missouri, USA) in PBS for one hour. The sections were incubated with the primary antibody anti-CaMKII, 1:200 (Millipore, Massachusetts, USA) in antibody diluent solution (Invitrogen, New York, USA) overnight. Sections were washed three times, for 15 minutes each with PBS, and incubated with the secondary antibody Alexa 488 anti-rat, 1:200 (Life Technologies, New York, USA) for three hours. Images were acquired using a Zeiss microscope (Axio Imager, Carl Zeiss, Inc., New York, USA) and then quantitatively analyzed with ImageJ software (NIH).

Immunohistochemical staining was performed using Envision Plus system and DAB kit. Briefly, the 4 μm tissue sections were deparaffinized using xylene, dehydrated in an ethanol gradient, and then rehydrated with deionized water. The sections were autoclaved in 1 mM EDTA buffer (pH 8.0) at 120°C for 2 min and cooled to 30°C. The nonspecific sites in the slides were blocked using 10% normal calf serum in phosphate-buffered saline (PBS) for 10 min. Next, an anti-EphA7 polyclonal antibody (Abgent, San Diego, CA, USA) at a 1 : 600 dilution in antibody diluent solution (Zymed, Invitrogen, Carlsbad, CA, USA) was dropped onto the slides and incubated at 4°C overnight. Following incubation, the slides were washed with PBS, stained with 3.3′-diaminobenzidine, and counterstained with hematoxylin. EphA7 expression was assessed as positive when the cytoplasm was stained brown. The immunoreactivity of EphA7 was evaluated independently by two pathologists. Any differences in results were verified by consensus.

Human breast tissues, provided by Seoul National University Hospital with approval from the SNUH Institutional Review Board (IRB; IRB No. 1904–141-1029), were fixed in 4% paraformaldehyde for 24 h at 4 °C and embedded in paraffin. The paraffin blocks were cut into 4-μm-thick sections, and the paraffin slides were deparaffinized with xylene and hydrated with graded ethanol. Antigen was unmasked by boiling the sections in 100 mM citrate buffer (pH 6.0) for 10 min and endogenous peroxidase activity was blocked with 3% hydrogen peroxide for 10 min. The slides were blocked in 5% normal goat serum (Vector Laboratories, Burlingame, CA, USA) for 1 h and incubated with primary antibodies (diluted 1:100–200 in antibody diluent solution; Life Technologies, Carlsbad, CA, USA) overnight at 4 °C. The following day, sections were incubated with biotin-conjugated secondary antibodies (diluted 1:100–200 in antibody diluent solution; Vector Laboratories) for 1 h and incubated with avidin-biotin complex reagent for 1 h. The slides were visualized using DAB staining solution (Dako, Santa Clara, CA, USA) followed by counterstaining with hematoxylin solution (Dako). Images of stained sections were obtained using the LAS microscope (Leica Microsystem, Wetzlar, Germany).

After permeabilization of the explanted tissues with 0.5% Triton X-100 (cat.# 9002-93-1, Sigma Aldrich, Germany) in PBS, the specimens were incubated with 4% blocking solution (goat serum cat.# 005-000-121, Jackson ImmunoResearch Europe Ltd., Ely, UK) for 60 min at room temperature (RT) to block non-specific binding sites. The explants were then incubated with the primary antibody, mouse monoclonal anti-neurofilament-200 (cat. # N0142, Sigma-Aldrich, Darmstadt, Germany, dilution 1:400) for 40 min at RT and then rinsed with PBS. The secondary antibody was the goat anti-mouse IgG (H + L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 488 (cat. # A 11001, Thermo Fisher Scientific, Karlsruhe, Germany; dilution 1:400) prepared in Antibody Diluent Solution (cat. # ab64211, Cell Signaling Technology Europe, Frankfurt am Main, Germany). The specimens were incubated with the antibodies for 60 min at RT.