Anti lc3

Four 5 μm sections from different areas of the brain and eye were stained by periodic acid Schiff hematoxylin (PASH) or hematoxylin and eosin stain respectively. Histopathologic changes (Brain: microglial nodules, perivascular and diffuse inflammation; Retina: disruption of architecture, perivascular and vitreal inflammation) were scored from 0 to 4 similar to previously described criteria^{36 (link),37 (link)}. Sections were also incubated with anti-T. gondii Ab (BioGenex) and Tomato lectin-DyLight 488 (Vector laboratories) that efficiently labels neural endothelial cells^{38 (link)} (at 0.5 μg/ml it stains neural endothelial cells rather than microglia) or anti-CD31 Ab (Elabscience). Coronal sections at the septo-diencephalic region (level of the thalamus) were examined at X400. The numbers of clusters of T. gondii parasites within tomato lectin⁺ elongated structures (endothelial cells) were counted per whole coronal section. Brain sections were also stained with anti-LC3 (Abgent) or anti-LAMP-1 (Developmental Studies Hybridoma Bank) Abs. Accumulation of LC3 or LAMP-1 around T. gondii located in endothelial cells was defined as the presence of a ring-like structure that surrounds the parasite^{21 (link),22 (link)}. Slides were analyzed using Leica DMI 6000 B automated microscope equipped for epifluorescence microscopy.

Total cellular protein lysates were prepared by harvesting cells in protein extraction buffer for 1 h at 4 °C as described previously [32 (link)]. GAPDH was used as the protein loading control. Anti-PAI-1, anti-Bax and anti-Beclin 1 were obtained from Cell Signaling Technology (Ipswich, MA, USA); anti-HIF-1α was obtained from BD Transduction Laboratories (San Diego, CA, USA); anti-GAPDH was obtained from Abcam (Cambridge, MA, USA); anti-LC3 and anti-CTGF were obtained from Abgent (San Diego, CA, USA); anti-p62/SQSTM1 was obtained from MBL (Nagoya, Japan); anti-pro-caspase 3 and anti-cleaved-caspase 3 were obtained from Epitomics (Burlingame, CA, USA).

Sections (2-µm thick) from formalin-fixed, paraffin-embedded tumor nodules were stained with hematoxylin and eosin or with anti-human Ki-67 (Dako, Cernusco sul Naviglio, Milan, Italy), anti-LC3 (Abgent), and anti-SQSTM1 (Sigma) specific antibodies. Images were analyzed using Image-Pro Analyzer software. Tumor apoptosis and necrosis were detected by TUNEL staining (Roche Diagnostics GmbH). The sections were examined under a light microscope (IX51; Olympus). Image analysis was performed using the open source software ImageJ [38 (link)].