Tissuefaxs spectra systems

Immunohistochemistry (IHC) staining was performed using an IHC Kit (Sigma-Aldrich, St. Louis, MO, USA), according to the manufacturer’s instructions. Paraffin-embedded sections were prepared, mounted on silane-coated slides, deparaffinized in xylene, rehydrated in a graded alcohol series, and rinsed in deionized water. After antigen retrieval, intrinsic peroxidase activity was blocked by incubation with 3% H₂O₂. Non-specific antibody-binding sites were blocked using 3% BSA in PBS. Sections were then incubated with appropriately diluted primary antibodies at 4 °C overnight. Secondary antibody (biotin-labeled goat anti-rabbit IgG) was applied for 1 h at room temperature. Staining was detected with 3,3ʹ-diaminobenzidine tetrahydrochloride (DAB) and observed under a Leica microscope (Leica microsystems, Wetzlar, Germany). For dystrophin staining, after incubation with primary antibody, the sections were incubated with goat anti-rabbit IgG (H + L) cross-adsorbed secondary antibody Alexa-Fluor^® 594 conjugated (Thermo Fisher Scientific, Hemel Hempstead, UK). The dystrophin immunofluorescence-stained sections were then examined with TissueFAXS^® Spectra systems (TissueGnostics, Vienna, Austria). ImageJ software was used to quantify dystrophin-positive staining and IHC staining through a cross-sectional area of muscle fibers and densitometry, respectively.

We collected pathologically confirmed pancreatic cancer specimens from 20 patients undergoing radical pancreatic cancer surgery from the Peking Union Medical College Hospital (PUMCH) with the approval of the Institutional Ethics Committee of PUMCH (Ethic code: I-22PJ487). Twenty surgical pancreatic cancer specimens were fixed in formalin and then embedded in paraffin. Multispectral IF staining was performed as previously described^{62 (link)}. These tumor tissue sections were incubated with the following antibodies: anti-CD66b (Abcam, ab207718, dilute at 1:500), anti-Myeloperoxidase (Abcam, ab208670, dilute at 1:100), anti-CD68 (Abcam, ab213363, dilute at 1:100), and anti-LC3B (Abcam, ab192890, dilute to 1 µg/ml). Nuclear staining was performed with ProLong Diamond Antifade mounting medium containing DAPI (Invitrogen). Images of tissue specimens were obtained using the TissueFAXS Spectra Systems (TissueGnostics GmbH, Vienna Austria). With the help of StrataQuest analysis software (Version 7.1.129, TissueGnostics GmbH, Vienna, Austria), we separated the multi spectral image (5-color staining) and set a threshold to divide the positive cells for analysis of cell number and location distribution.