Murine spleens were fixed in 10% neutral buffered formalin (for 48 h) and then dehydrated and embedded in Paraffine (Leica ASP 300S) according to routine methods. To detect and analyze infiltration with neoplastic cells, blocks were cut (2 μm thickness) and stained with Hematoxylin-Eosin or anti-human CD20cy antibody (Agilent, clone L26, 1:2000). CD20 IHC was performed on a Leica BondRxm using a Polymer Refine detection kit. All slides were scanned with a Leica AT2 scanning system with ×40 magnification. HE stainings and IHCs were evaluated by a board-certified veterinary pathologist and the amount of CD20 positive neoplastic cells in the spleen was analyzed by using the Aperio PositivePixelCount v9. The number of positive pixels was calculated per mm2 and the results between the groups (3AC-treated vs. control) were visualized and statistics (Mann–Whitney U test) were calculated with IBM SPSS statistics 25.
At2 scanning system
Manufactured by Leica
Sourced in Germany
The AT2 scanning system is a microscope-based digital imaging solution designed for high-resolution scan acquisition. The core function of the AT2 system is to capture detailed images of samples with a high degree of precision and clarity.
Automatically generated - may contain errors
Lab products found in correlation
Bond rxm, by Leica (2 mentions)
Asp300, by Leica (1 mentions)
Spss statistics 25, by IBM (1 mentions)
β3 tubulin, by Cell Signaling Technology (1 mentions)
Collagen 1, by Thermo Fisher Scientific (1 mentions)
Il 1β, by Abcam (1 mentions)
Tnf α, by Affinity Biosciences (1 mentions)
Cd206, by Abcam (1 mentions)
α sma, by Proteintech (1 mentions)
Polymer refine detection kit, by Leica (1 mentions)
Bond rxm system, by Leica (1 mentions)
A0398, by Agilent Technologies (1 mentions)
4 protocols using at2 scanning system
1
Murine Spleen Immunohistochemical Analysis
2
Comprehensive Histological Analysis of Bone
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Following micro-CT test, the specimens were prepared (decalcified, dehydrated, paraffin-embedded, and sectioned). H&E and Masson's trichrome staining were performed. Immunohistochemical (IHC) staining was performed to detect CD206 (1:1000, Abcam), iNOS (1:1000, Abcam), CD31 (1:5000, Abcam), α-SMA (1:1000, Proteintech), TNF-α (1:100, Affinity), and IL-1β (1:200, Abcam). Tissue slices were captured using an Aperio AT2 scanning system (Leica, Germany). Immunofluorescence staining was performed using CGRP (1:100, Abcam), β3-Tubulin (1:200, Cell Signaling Technology), OCN (1:100, Santa Cruz Biotechnology) and collagen I (1:500, Thermo Fisher). Images were taken using CLSM. The quantitative analysis was performed using Image J software.
3
Kidney Histopathology and Apoptosis Quantification
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Kidneys were fixed in 4% formalin in PBS and embedded in paraffin. Sections of 2 μm were cut. Renal lesions were scored on sections stained with Hematoxylin-Eosin and PAS according to standard protocols. Cleaved Caspase 3 immunohistochemistry was performed on a BondRxm system (Leica, Wetzlar, Germany). Briefly, the slides were deparaffinized, and heat-mediated retrieval (ER1) was applied for 20 min. A primary antibody against cleaved Caspase 3 was used (1:150, Cell Signaling, Danvers, cat. No. 9664), and binding was visualized using a Polymer Refine Detection Kit (Leica, Wetzlar, Germany) without postprimary antibody. All slides were scanned with an AT2 scanning system (Leica, Wetzlar, Germany).
Renal tubular injury was scored by the percentage of injured tubules with loss of brush border, cell lysis, and cast formation: 0, no damage; 1, <25%; 2, 25 to 50%; 3, 50 to 75%; 4, >75% (60 (link)). Cleaved Caspase 3 positive tubular epithelial cells were counted in 10 high power fields (40×) and averaged.
Renal tubular injury was scored by the percentage of injured tubules with loss of brush border, cell lysis, and cast formation: 0, no damage; 1, <25%; 2, 25 to 50%; 3, 50 to 75%; 4, >75% (60 (link)). Cleaved Caspase 3 positive tubular epithelial cells were counted in 10 high power fields (40×) and averaged.
4
Immunohistochemistry Protocol for Lymphoid Neoplasms
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Immunohistochemistry (IHC) was performed on a Bond Rxm (Leica) using a Polymer Refine detection kit without post-primary antibody. Slides were deparaffinized and pretreated with Epitope retrieval solution 1 (ER1, citrate buffer, pH = 6) or solution 2 (ER2, EDTA buffer, pH = 9) as indicated. The following primary antibodies were used: rat anti-B220/CD45R (B220, BD Bioscience, 1:50 dilution, ER1, 20 min), rat anti-CD138 (281-2, BD Bioscience, 1:59, ER2, 20 min), rat anti-MPO (A0398, DAKO, 1:100, ER2, 20 min), rabbit anti-CD3 (Sp7, DCS, 1:100 dilution, ER1, 20 min), rabbit anti-Tdt (005, Supertechs, 1:100, ER2, 20 min) and rat anti-CD4 (GHH4, Dianova DIA-404, 1:50 dilution, ER2, 40 min). Rabbit anti-rat secondary antibody (Vector, 1:400) was applied for primary rat antibodies. Slides were counterstained with hematoxylin and coverslipped after manual rehydration. Slides were scanned with a Leica AT2 scanning system. HE stainings and IHCs were evaluated by experienced mouse pathologists, who were blinded to the mouse genotypes according to the Bethesda proposals for classification of lymphoid neoplasms.89 (link)
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