Normal horse serum

Primary mixed glia and primary microglia isolates were generated as previously described (Zhu et al., 2016 (link)). Mixed glial cultures were determined to be 1–3% microglia and 97–99% astrocytes by composition. For further studies we used the spontaneously immortalized murine microglial cell line SIM-A9, which were provided generously as a gift by Dr. Kumi Nagamoto-Combs (Nagamoto-Combs et al., 2014 (link)). SIM-A9 were maintained in DMEM/F12 supplemented with 10% heat-inactivated FBS, 5% normal horse serum, and 50 U/mL penicillin/streptomycin (Gibco), with incubators maintained at 37°C and 5% CO2.

Formalin-fixed, paraffin-embedded brain sections were assayed for SIV viral RNA expression by in situ hybridization (ISH). Briefly, deparaffinized and rehydrated sections were first incubated with pre-hybridization buffer at 50 °C for 30 min to avoid background staining. Tissue sections were hybridized overnight at 50 °C with either sense or antisense digoxigenin-UTP-labeled SIVmac239 probe that spanned the entire genome. The hybridized sections were then blocked with 2 % normal horse serum (Gibco) and 1 % bovine serum albumin (BSA) in 0.1 M Tris/HCl (pH 7.4) for 1 h and incubated with sheep anti-digoxigenin-alkaline phosphatase (Roche Molecular Biochemicals) at 37 °C for 30 min, followed by staining with BCIP/NBT (Sigma) and counter staining with methyl green. ISH-stained sections were visualized and photographed with a camera-assisted microscope (OLYMPUS, BX53).

To investigate the antibody distribution within tumor tissues, 61B-Cy5.5 or hIgG-Cy5.5 (0.2 nmol) was injected into each mice bearing U87MG or HT29 tumor via tail vein. At 48 h after injection, the mice were euthanized, and the tumors were dissected, embedded in Tissue-Tec optimal-cutting-temperature compound (Sakura Finetek, Torrance, CA, USA) and cut into 8 μm sections. Frozen sections were fixed in ice-cold acetone for 5 min, blocked with 10% normal horse serum (Gibco, Grand Island, NY, USA) for 20 min, and then incubated with primary anti-CD31 antibody at room temperature for 30 min. After PBS buffer washing, the sections were incubated with secondary antibody for 30 min at room temperature. Subsequently, the slides were covered with EverBrite Mounting Medium containing DAPI and observed under Zeiss LSM 710 laser scanning microscope.

MACS-purified mouse SCs from approximately 20 dissociated sciatic nerve preparations were seeded in PLL/laminin-coated 35 mm dishes and cultured for ~20h in defined medium (10% Normal horse Serum (Gibco, #26050-088) in DMEM/F12, 350μg/mL BSA (Sigma, A4161), 1μM insulin (Sigma, I6634)) in preparation for ErbB2 receptor activation with recombinant Nrg1 according to a previously published method⁷¹ . SCs were subsequently control-treated or treated with 200 ng/ml recombinant Nrg1 (R&D Systems, 396-HB-050) for 24h, collected in RIPA buffer containing phosphatase and protease inhibitors, and then processed for protein analysis and western blotting using standard procedures. The SC preparations were randomly assigned to the control-treated and Nrg1-treated groups. Following primary antibodies were used: Phospho-ErbB2 Y1248 (1:500, Abcam, ab47755), Phospho-HER2/ErbB2 (Tyr877) (1:1000, Cell Signaling, 2241), PFKFB3 D7H4Q (1:1000, Cell Signaling, 13123), LDHA/LDHC C28H7 (1:1000, Cell Signaling, 3558), beta-actin Clone AC-74 (1:5000, Sigma, A2228).