Cs701

Mouse kidneys were snap-frozen in liquid nitrogen and embedded in OCT (Tissue-Tek) for preparation of 10-μm cryosections. For tissue visualization, frozen tissue sections were fixed with pre-chilled methanol on the Visium Tissue Optimization Slides (10X Genomics, PN-1000193), followed by staining with hematoxylin (S3309, Dako) and eosin (CS701, Dako). Brightfield histological images were taken with a Leica DMI8 whole-slide scanner. For RNA isolation and reverse transcription, sections were permeabilized with permeabilization enzymes to release mRNA, which was captured by probes on the Visium spatial gene expression slides (PN-1000184,10X Genomics). The captured mRNA was reversely transcribed to cDNA, spatially barcoded, amplified, and subjected to library construction using the Visium Spatial Library Construction Kit (PN-1000184,10X Genomics). Briefly, 10 μl of amplified cDNA from each sample was taken for library preparation through the processes of fragmentation, adapter ligation, PCR, and purification. The constructed library was sequenced with an Illumina Novaseq6000 sequencer with a sequencing depth of at least 100,000 reads per spot (performed by CapitalBio Technology, Beijing).

The bladder tissues (6.5 mm × 6.5 mm × 6.5 mm) from all participants were snap-frozen with precooled isopentane and then embedded with O.C.T. compound. Cryosections were cut with 10 μm thickness and mounted onto the GEX arrays. Sections were placed on Thermocycler Adaptor with the active surface facing up and incubated 1 min at 37 °C, and then were fixed for 30 min with methyl alcohol in −20 °C followed by staining with H&E (Eosin, Dako CS701, Hematoxylin Dako S3309, bluing buffer CS702).^{54 (link)} The brightfield images were taken via a Leica DMI8 whole-slide scanner at ×10 resolution.

The obtained BALF samples from lung cancer-suspected patients were performed via both the present dual-layer PERFECT filter system and the cytocentrifuge. Loading volume and filtration time for each sample was recorded. The collected cells on filters and glass slides were air dried and fixed with 95% ethanol for 10 min, followed by the hematoxylin-eosin (HE) staining. The procedure of HE staining included sequential immersions in hematoxylin (CS700, Dako, USA) for 2-3 min, bluing buffer (CS702, Dako, USA) for 10-15 s and eosin (CS701, Dako, USA) for 10-15 s. Rinse with distilled water was conducted after each immersion. Then they were dehydrated with 70% ethanol, 95% ethanol, absolute ethanol and dimethylbenzene sequentially, and mounted with neutral balsam for long-term stable storage.