Axiolab 5 microscope

Kidney grafts were harvested at designated time points and either immediately submerged in 10% formalin for paraffin embedding or preserved in OCT; and 4 μm sections were used for HE staining. IHC and immunofluorescence (IF) staining were performed as published previously (38 (link)). The Abs used included anti-CD3(Cell Signaling Technology, D4V8L), B220 (BioLegend, RA3-6B2), and C4d (Novus Biologicals, 16D2). To test the ability of DSA produced from single-cell cultures to bind to alloantigens, supernatants from the highest 5 DSA-positive clones were pooled and used to bind allogeneic native Balb/c kidney sections for 24 hours at 4°C. Then, primary Ab anti-IgG (SouthernBiotech) was used to detect the IgG. Images were captured on a ZEISS Axiolab 5 microscope.

Femora were fixed using formaldehyde 4% and embedded in paraffin blocks. After sectioning, samples were deparaffinized, hydrated, and stained in Alcian blue and Picrosirius red solutions [27 (link)]. Samples (n = 4) were observed using a Zeiss Axiolab 5 microscope and images were captured using an Axiocam 5 Colour Camera (Zeiss).

4 μm sections from formalin-fixed paraffin embedded (FFPE) mouse tumors were obtained. Tumor sections were deparaffinized, hydrated and antigen retrieval was performed by heating them in 10 mM citric acid (pH 6.0) for 10 min. SenTraGor™ reagent was applied as described in the SenTraGor™ staining section below. Blocking of non-specific binding of antibodies was performed by applying normal goat serum for 1 h at RT (dilution 1:40, Abcam ab138478). Then primary anti-biotin antibody (dilution 1:300, Abcam ab201341) and primary anti-Ki67 (dilution 1:200, Abcam ab16667) were applied and the sections were incubated overnight at 4 ^◦C. Development of positive signal was performed using the Dako REAL EnVision Detection System, (Cat.no: K5007) according to the manufacturer’s instructions. Tumor sections were observed using a ZEISS Axiolab5 microscope on a 20 × Objective (200 × magnification).

The following devices and softwares were used: (1) Zeiss Axiolab 5 microscope with Axiocam 305 colour camera for immunohistochemistry (Zeiss ZEN v3.7), (2) Leica TCS SP8 confocal laser scanning microscope (LAS X v.5.7.23225), (3) Zeiss Supra 40 Field Emission Scanning Electron Microscope to acquire transmission electron microscope (Zeiss SmartSEM v6.0) and 3D Modeling (3DMOD v4.9.10), (4) XF96 and X24 Seahorse analyzers (Agilent Technologies) to collect OCRs (WAVE Pro v10.0.1.84; v2.6.1.56, respectively), (5) Synergy HTX (BioTek) multi-mode plate reader (Gen5 v3.12), (6) StepOnePlus Real-Time PCR System (Applied Biosystems) for mRNA expression (StepOne v2.3), (7) Microsoft Excel v16.48, Microsoft Word v16.48, Microsoft PowerPoint v16.47, (8) Prism v8.4.3, (9) Endnote X9.3.3 and (10) ImageJ v2.0.0-rc-69/1.52p. Enrichment map was generated in Cytoscape v3.8.1, Enrichment map plugin v3.3.0. Handling and analyses of proteomics data were performed via Python v.3.7.4 and Scientific Python Stack: SciPy v.1.3.1, NumPy v.1.17.2 and Matplotlib v.3.1.1. Phosphosites showing significant regulation between groups were used to predict the kinase responsible for their catalysis using the iGPS software.

IMR90 fibroblasts were seeded on coverslips, fixed using 4% PFA diluted in PBS for 10 min at 4°C, and permeabilized by applying Triton‐X 0.3% in PBS for 15 min. Cells were washed with 1× PBS and incubated with 3% H₂0₂ for 18 min at RT in order to block endogenous peroxidase activity. The coverslips were washed with 1× PBS and incubated in goat serum for 1 h in RT (Abcam; ab138478) serving as blocking solution. Cells were subsequently incubated with primary anti‐Ki67 antibody (1/200, Abcam; ab16667) for 1 h at RT. Development of positive signal was carried out using the Dako REAL EnVision Detection System (K5007), according to the manufacturer's instructions. Cells were counterstained with hematoxylin, mounted and observed on a Zeiss Axiolab 5 microscope using the 20× objective.

After incubation, one of the triplicate tissue samples from each condition was washed with PBS, fixed in 10% formalin, embedded in paraffin, and sectioned by microtome and submitted to hematoxylin and eosin staining (H&E). Samples were examined under digital microscope (40× magnification, Axiolab 5 microscope with Axiocam camera and ZEN 2.0 software, Zeiss, Sartrouville, France). Each image was studied for morphological changes according to the International Harmonization of Nomenclature and Diagnostic Criteria, INHAND [26 (link)].