Superfrost plus charged slides

For the immunohistochemical experiments, mouse bowel tumors were fixed in 10% formalin for 24 h and were paraffin-embedded. The paraffin blocks were cut onto Superfrost Plus charged slides (Thermo Fisher Scientific, Waltham, MA, USA), deparaffinized in xylene, and hydrated with alcohol. The peroxidase activity was quenched with a 3% H₂O₂/methanol blocking solution for 30 min. The slides were boiled in 0.01 M sodium citrate pH 5.0 for 20 min to retrieve the antigens. Cells cultured on the chamber slides were fixed in acetone/methanol (1:1). For immunohistochemistry, all of the slides were blocked in avidin and biotin blocking solutions (Vector Laboratories, Burlingame, CA, USA). The slides were incubated with the primary antibodies overnight at 4 °C. After 3 washes in tris-buffered saline, the slides were incubated with a biotinylated secondary antibody (1:200). The slides were washed again with tris-buffered saline and then incubated with peroxidase-linked avidin using the Vectastain ABC kit (Vector Laboratories, Burlingame, CA, USA) for 30 min. The slides were rinsed in tris-buffered saline and stained with 3-3′-diaminobenzidine chromogen, then counterstained with hematoxylin. Appropriate negative controls for the immunostaining were prepared by omitting the primary antibody. Trichrome staining to detect collagen fibers was conducted as previously published [21 (link)].

IHC staining was performed on 30-μm sections mounted on Fischer Scientific Superfrost Plus charged slides in the brain of chronically survived cohort of mice using the standard protocol as described previously27 (link). BDNF, MBP and GFAP antigen were probed using their specific antibody [BDNF 1:500 (BD bioscience); Myelin basic protein (MBP) 1:1000 (Abcam) or GFAP-Cy3 1:500 (Sigma)]. Three coronal brain sections per mouse (n = 4 per group), taken 0.02, 0.45, and 0.98 mm from bregma, were stained and visualized for quantification at 20×/63× magnification at the core/penumbra junction (Supplementary Fig. 1). A blinded observer quantified BDNF and DAPI positive cells using Image J software (NIH). The average numbers of cells visualized from 3 adjacent regions at the core/penumbra junction were recorded for each mouse. Confocal microscopy (LSM710 Metaconfocal laser scanning microscope, Carl Zeiss Micro-Imaging) was performed to visualize MBP on brain tissue sections.

E15.5 embryos were collected and fixed for 24 h in 4% paraformaldehyde (PFA) at 4°C. Embryos were then washed three times in 1× PBS and brains were dissected for gross measurements. After measurements were completed, fixed brains were placed in 30% sucrose for 16-36 h at 4°C then frozen in Optimal Cutting Temperature Compound (OCT; Sakura, Torrance, CA). Tissue blocks were stored at −80°C until use. Serial coronal sections (16 μm) were cut using a cryostat, mounted on Superfrost^® Plus charged slides (ThermoFisher Scientific, Waltham, MA), and stored at −80°C.