Tissue slices used for DESI-MSI analysis were stained after completion of the MSI acquisition using a standard Hematoxylin and eosin (H&E) staining protocol. Sections were washed in successive EtOH baths (100%, 96%, 96%, 70%, 70%) and deionized H2O for 3 min each. Hematoxylin (Merck, Darmstadt, DE) staining was performed for 3 min followed by a gentle 3-min wash with running tap water. Eosin (Avantor Performance Materials B.V., Arnhem, NL) staining was executed for 30 s and washed gently with running tap water for 3 min. The staining was finalized by an EtOH wash for 1 min and a xylene wash for 30 s. The slides were covered by placing coverslips on the stained tissues using Entellan (Merck, Darmstadt, DE). Optical images were acquired using a VENTANA iScan HT scanner (Roche Diagnostics, Indianapolis, IN, USA).
Ventana iscan ht scanner
Manufactured by Roche
Sourced in United States
The VENTANA iScan HT scanner is a digital slide scanning system designed for high-throughput operation in pathology laboratories. It captures high-resolution digital images of tissue samples mounted on glass slides. The scanner provides rapid and efficient digitization of slides to support diagnostic and research workflows.
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3 protocols using ventana iscan ht scanner
1
H&E Staining for DESI-MSI Analysis
2
Histological Tissue Staining Protocols
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For hematoxylin and eosin (H&E) staining, tissues were harvested and fixed immediately in 10% neutral buffered formalin. Dehydration, clear and paraffinization were performed on a Tissue-Tek VIP Vacuum Infiltration Processor (Sakura). The samples were embedded in paraffin using a Tissue-Tek TEC Tissue Embedding Station (Sakura) and sectioned at 5 μm; the sections were placed on positively charged glass slides. The slides were deparaffinized, rehydrated and stained with Modified Mayer's Hematoxylin and Eosin Y Stain (America MasterTech Scientific) on a H&E Auto Stainer (Prisma Plus Auto Stainer, Sakura) following standard laboratory procedures.
For Masson's trichrome staining, staining was conducted using the Epredia™ Richard-Allen Scientific Masson Trichrome Kit (Thermo Fisher Scientific). Briefly, after deparaffinization and rehydration, the slides were immersed in Bouin's solution at 60 °C for 1 h. The slides were washed and stained in Weigert's hematoxylin. Next, the slides were stained in Biebrich scarlet-acid fuchsin and incubated in phosphotungstic-phosphomolybdic acid after staining with aniline blue. The slides were then rinsed, dehydrated and mounted. After H&E or trichrome staining, whole slide images were acquired with a Ventana iScan HT Scanner (Roche) and viewed by iScan image viewer software.
For Masson's trichrome staining, staining was conducted using the Epredia™ Richard-Allen Scientific Masson Trichrome Kit (Thermo Fisher Scientific). Briefly, after deparaffinization and rehydration, the slides were immersed in Bouin's solution at 60 °C for 1 h. The slides were washed and stained in Weigert's hematoxylin. Next, the slides were stained in Biebrich scarlet-acid fuchsin and incubated in phosphotungstic-phosphomolybdic acid after staining with aniline blue. The slides were then rinsed, dehydrated and mounted. After H&E or trichrome staining, whole slide images were acquired with a Ventana iScan HT Scanner (Roche) and viewed by iScan image viewer software.
3
Inflammatory Response in Craniotomy Infection
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TLR2 KO, caspase-1 KO, and WT animals were deeply anesthetized using sodium pentobarbital and transcardially perfused with PBS followed by 4% paraformaldehyde (PFA). To preserve the inflammatory response in the galea, the entire head was collected and post-fixed in 4% PFA for 4 days. The head was decalcified in 20% ethylenediaminetetraacetic acid (EDTA) for 14 days and cryoprotected with 30% sucrose for 4 days prior to embedding in optimal cutting temperature medium. Cryostat sections (16 μm) were collected every 100 μm and mounted on SuperFrost slides (ThermoFisher), and representative tissue sections encompassing the craniotomy infection site were rehydrated and subjected to H&E staining. Stained sections were imaged on a Ventana iScan HT scanner (Roche, Indianapolis, IN) and are presented at × 2 magnification to demonstrate the extent of infection involvement.
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