Lf200 microscope

IHC staining was carried out as previously described [15 (link)]. In brief, tissue Sections (4 μm) were dewaxed in xylene twice for 2 min each and then rehydrated in a graded series of ethanol (100–70%). Antigen retrieval was performed by boiling sections for 15 min in sodium citrate buffer (10 mM citrate acid, 10 mM sodium citrate, pH 6.0). Then, 5% normal donkey serum was used to block nonspecific antigens. IHC was performed using the Dako EnvisionTM method for antibody incubation and then developed by using the DAB peroxidase substrate kit (Beyotime, P0202). IHC-stained sections were imaged by a Leica LF200 microscope.
Antibodies for IHC included anti-S100 (Dako Z0311, 1:200), anti-Calponin (Dako M3556, 1:100), anti-SMA (Dako M0851, 1:100), anti-CK7 (Dako M7018, 1:50) and anti-CK19 (Dako M0888, 1:100), anti-FZD2 (Bioworld BS3163, 1:50), which were purchased from commercial sources.

All IMPA tissues and adjacent normal controls were fixed in 4% PFA for  > 4 h at 4 °C. Then, these fixed tissues were dehydrated with graded ethanol (70–100%) and embedded in paraffin. Sections (4 μm) were cut on a Leica HistoCore BIOCUT RM2235. Hematoxylin–eosin (H&E) staining was performed following standard procedures. Stained sections were imaged using a Leica LF200 microscope.

Immunohistochemistry (IHC) was performed on paraffin-embedded human glioma and normal brain tissues collected from Shanghai Ninth People’s Hospital. The sections were deparaffinized in a xylene gradient and rehydrated in an ethanol gradient. Antigen retrieval was performed in sodium citrate buffer (10 mM sodium citrate pH 6.0) at 100 C for 20 min. Then, endogenous peroxidase was deactivated by applying 3% H₂O₂ in methanol. IHC of SYDE1 was performed by the Dako EnvisionTM method. Briefly, the sections (3 μm) were sequentially incubated with the anti-SYDE1 antibody (NBP1-89350, Novus Biologicals) and the HRP-conjugated secondary antibody (ab6721, Abcam). Then, the sections were color-developed with a DAB Immunohistochemistry Color Development Kit (E670033, Sangon Biotech) and counterstained with hematoxylin.
A Leica LF200 microscope was used to image the stained sections. The intensity of SYDE1 signals was scored as negative (0), weak (1) or strong (2). The staining extent of SYDE1 was evaluated according to the immunoreactive tumor cell percentage, which was scored as I (0%, score = 0), II (1–5%, score = 1), III (6–25%, score = 2), IV (26–75%, score = 3) and V (76–100%, score = 4). SYDE1 signals (ranging from 0 to 8) were calculated by multiplying the intensity score with the staining extent score and were classified into low (0–4) or high (5–8) groups for Fisher’s exact tests.