Imagescope

An automated immunohistochemical staining system (BenchMark XT, USA) was used to perform immunohistochemical staining. Stained tissue microarray slides were scanned with a Leica Aperio AT2 scanner at 400× magnification. Digital images were analyzed by Leica Aperio ImageScope software with the Nuclear v9 algorithm. The following biomarkers were recorded: 1) IDH1 (isocitrate dehydrogenase 1), 2) 1p19q, 3) TP53, 4) ATRX, and 5) Ki67. IDH1 and 1p19q codeletion and alpha thalassemia/mental retardation syndrome X-linked (ATRX) status were scored as positive or negative. TP53 status was quantified as the percentage of stained nuclei: less than 10% of stained nuclei indicate an absence of immunoreactivity; 10-30% indicate a score of 1+; 30.1%–50% indicate a score of 2+; and more than 50% indicate a score of 3+. Scores of –1 or 1+ were regarded as TP53 negative, and 2+ and 3+ were regarded as TP53 positive. A Ki67 index was also calculated according to the percentage of Ki67-positive tumor cells present in the sample.

Mouse embryos obtained on E18.5 were fixed for 48 h in 4% formaldehyde in phosphate-buffered saline (PBS), transferred to sucrose, and embedded in paraffin. Serial sections (5 μm) were cut along the sagittal axis of the embryo. Slides were deparaffinised and rehydrated using xylene and graded ethanol. Antigen retrieval was performed in a pressure cooker at 7.5 psi in 0.1 M Tris-HCl buffer (pH 9.0) for 15 min. Slides were rinsed with water 6 times at room temperature and washed for 5 min in PBS. Endogenous peroxidase activity was quenched by incubation in 3% hydrogen peroxide in PBS for 30 min at room temperature. Slides were incubated for 16–18 h at 4 °C with primary anti-Flavivirus Group Antigen (Millipore, #MAB10216) diluted 1:250 in Dako Antibody Diluent with Background Reducing Components (Agilent, #S3022). After rinsing in PBS 3 times for 5 min each, the slides were incubated with a horseradish peroxidase-conjugated goat anti-mouse secondary antibody (Abcam, #ab2891) for 30 min at room temperature. Slides were washed again in PBS, incubated for 3 min with DAB complex (ImmPACT DAB Peroxidase Substrate, Vector Laboratories, #SK-4105), and washed 3 times each in PBS and water. Finally, slides were counterstained in Mayer’s hematoxylin, mounted, visualised, and recorded using an Aperio automated system (Leica). The images were analysed using ImageScope (Leica).

ISH assay was used to detect the HAGLORS expression in the bladder tissue microarray. The HAGLROS probes were designed and synthesized by Boster Biological Technology (Wuhan, China). The images were analysed by ImageScope (Leica, Mannheim, Germany) software and the expression level of HAGLORS was evaluated by histochemistry score (H-Score).

Hemiforebrains were immersion fixed for 48 hours in 10% neutral buffered formalin in 4°C, blocked coronally into four 3mm slabs using a brain matrix, and processed and embedded into paraffin. Immunohistochemistry was performed on 5 μm coronal sections using automated immunostainers (Ventana Medical Systems, Discovery XT) with nY-29 (Covance, 1:200) mouse monoclonal antibody and counterstained with hematoxylin. After coverslipping, slides were digitized using a slide scanner (Aperio ScanScopeXT, Leica) and numbers of NFT positive neurons were counted in the entorhinal cortex region using image analysis software (Aperio ImageScope) after manual determination of the region of interest.