Positively charged glass slides

Briefly, 6-μm sections of knee joint tissue were placed on positively charged glass slides (Thermo Fisher Scientific, Asheville, NC, USA) and then dried with a hotplate to increase the adherence of the tissues to the slides. After the samples were deparaffinized and rehydrated according to conventional methods, endogenous peroxidases were blocked with 3% hydrogen peroxide in methanol (Sigma-Aldrich, Burlington, USA). After 30 min the samples were digested with 5 mg/ml hyaluronidase in phosphate buffered saline (PBS) (Sigma-Aldrich) for 20 min and then were incubated with anti-CD31 (1:100 dilution) (SC-56; Santa Cruz, CA, USA) and anti-CD34 (1:100 dilution) (Santa Cruz, CA, USA) antibodies overnight at 4 °C. Following treatment of the samples with appropriate biotinylated secondary antibodies, a 3,3’diaminobenzidine (DAB) streptavidin-peroxidase (SP) DAB Histostain-SP immunohistochemistry kit (ZYMED203 Laboratories/Invitrogen, Carlsbad, CA, USA) was used to visualize bound antibodies. After the sections were counterstained with hematoxylin (ZYMED Laboratories/Invitrogen), the tissues were imaged with a Nikon Ri 1 microscope (Nikon, Melville, NY, USA) (Guan et al. 2012 (link)).

For IHC staining, paraffin-embedded tissues were sectioned, mounted on positively charged glass slides (Thermo Scientific, MA, USA), baked, deparaffinized and rehydrated. Antigen retrieval was completed by heating slides in citrate buffer (10 mmol/L; pH 6.0) for 20 min. Sections were incubated overnight at 4°C with mouse monoclonal antibody Notch1 (1:300) or rabbit polyclonal antibody NR4A2 (Nurr1) (1:200) and then washed with PBS. The washed sections were incubated respectively with HRP-conjugated goat anti-rabbit IgG secondary antibodies and goat anti-mouse IgG secondary antibodies (EarthOx, CA, USA) at room temperature for 30 min. and visualized with an Eclipse Ci-s (Nikon, Japan).

Eight micrometres transverse sections of frozen tibialis anterior and brain tissues were prepared using a cryostat (CM 1900; Leica) air‐dried for 15 min. and stored on positively charged glass slides (Thermo Scientific, Waltham, Massachusetts, USA) at −80°C. When needed, sections were thawed, fixed in 4% formaldehyde, blocked using 0.1% gelatin in PBS for 30 min. at room temperature (RT) and then incubated at RT for 1 hr with primary antibodies, washed with PBS‐gelatin and incubated with Alexa‐conjugated secondary antibodies. Sections were washed and finally mounted with Mowiol or PBS‐glycerol, pH 8.0, containing 1% n‐propyl gallate. In the case of NMO‐IgG staining, tissue sections were only fixed at the end of the staining procedure to preserve conformational epitope integrity as previously reported 36.

Tumor tissues derived from nude mice were fixed in formalin solution for more than 24 h. For IHC staining, tissues were sectioned (4 μm), mounted on positively charged glass slides (Thermo Scientific, MA, USA), baked, deparaffinized and rehydrated as previously described [14 (link)]. Antigen retrieval was completed by heating slides in citrate buffer (10 mmol/L; pH 6.0) for 20 min in a pressure cooker. Sections were incubated overnight at 4°C with GP73 rabbit polyclonal antibody (1:1,000) and then washed with PBS. Washed sections were incubated with HRP-conjugated goat anti-rabbit IgG secondary antibodies (EarthOx, CA, USA) at room temperature for 30 min and visualized with 3, 3′-diaminobenzidine (Vector Laboratories, Burlingame, CA, USA.

To validate the localization of biotinylated proteins, biotinylated and non-biotinylated (control) worms were embedded and frozen in OCT compound (Tissue-Tek, Sakura). Ten µm cryosections were prepared, transferred onto positively charged glass slides (Thermo Fisher Scientific, Germany) and fixed in acetone for 30 min at − 20 °C. Next, the sections were hydrated for 10 min with PBS, washed with PBST (PBS with 0.05% Tween20) and blocked with 1% BSA (bovine serum albumin) in PBST (blocking buffer) for 1 h. For biotin detection, the sections were incubated with avidin-FITC conjugate (A821, Thermo Fisher Scientific, Germany) diluted in blocking buffer at a final concentration of 2 μg/ml for 1 h. After washing five times with PBST, the sections were mounted (Immunoselect Antifading Mounting Medium DAPI, DIANOVA, Germany) and evaluated using a fluorescent microscope (100×, Axio Scope.A1; Carl Zeiss Microscopy, Germany) equipped with an AxioCam MRc camera (Carl Zeiss Microscopy, Germany).