At2 scanner

RNAscope stained FFPE cell sections were scanned using Leica AT2 scanner (Leica, US). Whole sections were examined at 40× magnification. RNAscope results of HPV were recorded based on signal location and positive cells. For probe-HPV-HR and probe-HPV-LR results, the signal locations of cells and the positive cell numbers in each sample, < 3, ≥3≤10 or > 10, were recorded. Besides the classic RNAscope dot signals detected in cytoplasm or nucleus, there were HPV RNA signals gathered as clusters, i.e. big amount of dot signals with high-density in limited area, above one or more cells which were recorded as well. For TERC results, RNA signals were only discovered in nucleus which were recorded.
Two gynecologists (Z. H. and H. Y.) evaluated the scanned sections independently. If a disagreement occurred during RNAscope assay result recording, they reviewed the case together and reached a final agreement. The interpretation was generally straightforward; therefore, no significant disagreements led to incompatibility.

Livers and kidneys were fixed in 10% normal buffered formalin for 24 h, washed and stored in 70% ethanol until paraffin embedding and sectioning. Sections were stained by hematoxylin and eosin (H&E) and Picrosirius Red, according to standard protocols (staining performed by Histoserv, Inc.). Engrafted hepatocytes were detected by IHC staining for FAH (ab151998) and human ASGR1 (ab254261), or RNAscope staining for human albumin (ACD, 457511). Liver zonation was observed by IHC for GS (ab125724), and viral transduction was monitored by GFP, by both IHC (ab183734) and RNAscope (EGFP probe, ACD 400281). All histology slides were scanned using a Leica Aperio AT2 scanner. FAH IHC staining was quantified using ImageJ.

Tumor section processing and TUNEL staining were performed by HistoWiz (Brooklyn, NY). Briefly, all IHC staining is automated using BOND Rx. Slides were treated with Dewax, a solvent-based solution (Leica Biosystems). Tissue sections were fixed by adding 10% neutral buffered formalin for 15 minutes. Slides were treated with Proteinase K at 1:500 dilution. Tissue sections were re-fixed using 10% neutral buffered formalin. Tissue sections were then treated with the equilibration buffer and incubated for 12 minutes at room temperature (RT). Subsequently the TdT reaction mix was added and incubated for 60 minutes at 37°C. The TUNEL reaction was stopped by adding 2X SSC. Tissue slides were treated with Peroxide Block (3-4% Hydrogen Peroxide) followed by wash buffer. Streptavidin HRP was added and incubated for 30 minutes at RT. DAB was added onto the slides and incubated for 10 minutes. Counter-staining was done by adding hematoxylin. Slides were covered using the Sakura Tissue Tek strainer and cover slips then scanned at 40x using the Leica AT2 scanner.

Immunohistochemical staining was performed on an automated immunohistochemical staining system (BenchMark XT, USA. 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-R132H (isocitrate dehydrogenase 1), 2) 1p19q, 3) P53, 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. P53 status was quantified as the percentage of stained nuclei: less than 10% of stained nuclei indicated an absence of immunoreactivity; 10–30% indicated a score of 1+; 30.1–50% indicated a score of 2+; and more than 50% indicated a score of 3+. Scores of − 1 or 1+ were regarded as P53 negative, and 2+ and 3+ were regarded as P53 positive. The Ki-67 index was also calculated according to the percentage of Ki-67 positive tumor cells present in the sample.

Immunostaining was performed as described previously^{24 (link)}. Mouse brains were cut into 35 µm sections on a horizontal sliding microtome. The free-floating sections were immune-blocked with 4% goat serum in 0.25% triton/PBS for 1 h. Dopaminergic neurons were stained with anti-tyrosine hydroxylase (TH, 1:4,000, overnight at 4 °C). Sections were incubated with biotinylated secondary antibody for 1 h followed by incubation with Vectastain ABC reagents (Vector Labs, Burlingame, CA) for 40 min and then color was developed with 3,3-diaminobenzidine. To monitor DA neuro degeneration, two individuals blind to the treatment counted the number of TH-immunoreactive (TH-IR) neurons in the SN pars compacta (SNpc) of six evenly spaced brain sections from a series of 24 sections that covered the entire SN^{26 (link)}. Stereological counts of THir SNpc neurons were estimated using an optical fractionator method on an Olympus BX50 stereological microscope within user-defined boundaries^{24 (link)}. All immunohistochemistry images were captured by a Leica Aperio AT2 Scanner.

Two 2.0-mm diameter samples were removed then transferred to a recipient paraffin block to construct TMAs using a Tissue Array MiniCore 3 (ALPHELYS, Plaisir, France). Samples (4-µm thick) were obtained from each TMA with Leica Rotary Microtome RM2135 (Wetzlar, Germany). Immunohistochemical staining was performed on Leica BOND III automated system. All samples were pretreated in a 65 °C oven for 1.5 h. Staining was performed with dewaxing and epitope retrieval. Samples were blocked with peroxide solution for 7 min. Selected antibodies (shown in Additional file 1: Table S1) were used to incubate samples, followed by post-primary for 8 min, polymer for 8 min, diaminobenzidine (DAB) for 5 min, and hematoxylin counterstain for 2 min. Finally, the samples were dehydrated, cleared, then fixed with neutral resins. Stained samples were scanned with Leica Aperio AT2 scanner (400× magnification) and analysed by two different pathologists. Samples were scored as negative (0+), weak (1+), moderate (2+), and strong (3+) signal. The percentage of positivity was also calculated by two pathologists. The histological scores (H-Score) was calculated using following formula: H-Score = 0 × (percentage of negative) + 1 × (percentage of weak) + 2 × (percentage of moderate) + 3 × (percentage of strong). Thus, the H-Score ranges from 0 to 300 [5 (link)].