Digital pathology slide scanner

All renal biopsy specimens were obtained by percutaneous needle biopsy and were routinely examined by light microscopy, immunofluorescence, and electron microscopy. Two experienced pathologists independently examined the kidney biopsy specimens. They had access to the patient’s clinical information, including vital signs, blood test results, urinalysis results, and clinical diagnoses. Pathological parameters such as activity index score (AIs) and chronicity index score (CIs) were evaluated as described previously (16 (link)).
Infiltrating macrophages and lymphocytes in renal tissue were identified using immunohistochemical staining. Paraffin-embedded tissue sections (2 µm) were incubated with the following primary antibodies: mouse anti-human CD68 mAb (M0876; DAKO, Glostrup, Denmark), rabbit antihuman CD3 antibody (SP7; LEICA), rabbit antihuman CD4 antibody (NCL-L-368; LEICA) and rabbit antihuman CD8 (NCL-L-295). The slides were then incubated with the secondary antibody and visualized using a LEICA System Kit and counterstained with hematoxylin. The stained sections were scanned with a Digital Pathology Slide Scanner (Leica, Wetzlar, Germany). Positive-stained cells were automatically counted in all nonglobally sclerotic glomeruli at 400 magnifications using Aperio eSlide Manager (Leica)(Figure 2).

Sample slides with 3-μm-thick tissue sections were deparaffined in xylene, rehydrated in descending concentrations of ethanol and PBS, and subjected to antigen retrieval by heated citrate buffer (pH 6.0). Endogenous peroxidases activities were blocked by 3% hydrogen peroxide. Slides were then incubated at 4°C overnight in the indicated antibodies, including those against MAPK6 (1:200; ab53277 from Abcam or sc-365234 from Santa Cruz Biotechnology) and p-AKT S473 (1:50, #3787 from CST or 1:150, AF11237 from AiFang Biological, China). IHC staining was then performed as previously described (30 (link)). IHC images were obtained on a Leica digital pathology slide scanner or a Leica microscope. IHC staining signal was evaluated on the basis of average optical density (AOD).

Lungs were harvested from mice infected with Mabc for 10 days. Lungs were fixed in 10% formalin and embedded in paraffin wax. For histopathology, lung paraffin sections (4 µm) were cut and stained for hematoxylin and eosin (H&E) as previously described [21 (link)]. For analysis of the extent of tissue necrosis, propidium iodide (PI) staining was performed. For analysis of the extent of tissue necrosis, lung paraffin sections (4 µm) were cut and immunostained with propidium iodide solution (P3566; Invitrogen, Carlsbad, CA, USA). After mounting, fluorescence images were acquired using a confocal laser-scanning microscope (LSM 710; Zeiss, CLSM, Jena, Germany), with constant excitation, emission, pinhole, and exposure-time parameters. H&E staining was scanned using an Aperio digital pathology slide scanner (Leica) and imaged using an Aperio ScanScope® CS System. To quantify the inflamed area and necrosis, the MFI of the red threshold was determined using FIJI software.

For lung metastases histology, 3 μm sections were cut from the lung lobes and stained with haematoxylin and eosin. For computer-aided assessment of lung tissue, the area occupied by metastatic foci, identifiable in sections of lung lobes, were calculated using a Leica Aperio Digital Pathology Slide Scanner and the software Leica Aperio Image Scope, summed and total value was finally compared to the whole area of the lung lobe.

Primary tumor, heart, lung, liver, spleen, and kidney tissues were fixed in 4% paraformaldehyde overnight and embedded in paraffin to obtain 5‐µm thick sections. All tissues were stained with hematoxylin and eosin (H&E) to visualize tumor progression, metastasis, and side effects. Primary tumor tissues were stained with TUNEL (Click‐iT Plus TUNEL Assay Kit, Alexa Fluor 594 dye, Invitrogen, USA) and Ki‐67 antibody (1:500, Novus Biologicals, catalog no. NB11089717F) to visualize tumor cell apoptosis and proliferation. Immunofluorescence images were acquired with an Axio Observer 7 inverted microscope (Zeiss, USA). Immunohistochemistry images were acquired with an Aperio Digital Pathology Slide Scanner (Leica, USA).

Tissue was soaked in 10% formaldehyde for 48 h at 4 °C. The paraffin section was done according to the usual procedure, and the thickness of the slice was 10 μm. Gradient alcohol was employed to dewax of sections before dyeing. The sections were stained with hematoxylin solution and eosin staining solution successively, dehydrated by gradient alcohol, and then putted in xylene for transparency. Then the sections were sealed with neutral resin. Images were acquired under a Aperio digital pathology slide scanner (Leica, Heerbrugg, Switzerland). Nanozoomer Digital Pathology (NDP) Image software was used to measure epidermal thickness. Five points in each section were measured, and the average of these points was taken as the final results of each sample.