Superfrost plus slides

Peripheral tissues were stored in 70% ethanol at 4 °C while brain tissues that were stored in PBS buffer at 4 °C were moved into ethanol 1 week prior to paraffin embedding. Dehydration in absolute alcohol (VWR chemicals) and xylene (Histolab) followed by paraffin (Histolab) immersion were performed using an automated Tissue Processing Center TPC 15 Duo (MEDITE) machine. After manual embedding into separate paraffin tissue blocks, one representative section (4um) was taken from each tissue block using microtome (Microm HM 355S, Thermo Fisher Scientific) with a microm STS Section-Transfer-System (waterfall) for section transfer into warm water bath (38 °C) stretching before placed on SuperFrost PlusTM slides (VWR). Slides were dried in room temperature for 24 h followed by 50 °C over night (LAMB Windsor Incubator E18.31, Histolab).

All peripheral pig tissues were stored in 70% ethanol at 4 °C. The pig brain tissues were stored in PBS buffer at 4 °C and changed into 70% ethanol one week prior to paraffin embedding. We first dehydrated the tissues with absolute alcohol (VWR chemicals) and xylene (Histolab). Next, paraffin (Histolab) immersion was performed using an automated Tissue Processing Center TPC 15 Duo (MEDITE) machine. After manual embedding into separate paraffin tissue blocks, one representative section (4um) was taken from each tissue block using a microtome (Microm HM 355 S, Thermo Fisher Scientific). A microm STS Section-Transfer-System (waterfall) was used for section transfer into a warm water bath (38 °C) stretching before placed on SuperFrost PlusTM slides (VWR). All slides were dried at room temperature for 24 h followed by 50 °C overnight (LAMB Windsor Incubator E18.31, Histolab).

Seedlings were fixed in 4% (v/v) paraformaldehyde (15710, Electron Microscopy Sciences, Hatfield, PA) with 5% DMSO in PBS for 30 min under vacuum. The fixed seedlings were quenched with three rinses in 50 mM NH₄Cl in PBS and then washed twice with PBS. Approximately 100 cotyledons were chopped with a razor blade on a slide in 50 μl lysis buffer (15 mM Tris-HCl pH 7.5, 50 mM EDTA, 0.5 mM spermine-4HCl, 80 mM KCl, 20 mM NaCl, and 0.1% Triton X-100). The resulting suspension containing the released nuclei was transferred into four volumes of nuclei suspension buffer (100 mM Tris-HCl pH 7.5, 50 mM KCl, 2 mM MgCl₂, 5% sucrose and 0.05% Tween-20). The nuclei suspension was divided into 30 μl aliquots, spotted on Superfrost® Plus slides (Avantor, Radnor, PA) and air-dried for 4 h at room temperature. The slides were either used immediately or stored at −20 °C.

A digoxigenin-labelled riboprobe was synthesized from linearized plasmid pCR4-TOPO (Invitrogen; nucleotides 2705–3558 of NM_001163847). Tissue samples were snap frozen in Optimal Cutting Temperature and 15 μm sections were cut using a cryostat (Leica) and mounted onto Superfrost Plus slides (Avantor). Probe hybridization, washing and signal detection using an alkaline phosphatase-conjugated anti-DIG antibody was carried out as previously described (20 (link)).

WT zebrafish eyes (~6 mpf) were enucleated and fixed in 4% paraformaldehyde/PBS at 4 °C overnight. After washing 3 times in PBS for 10 minutes, the eyes were incubated in 10%, 20% and 30% sucrose/PBS at 4 °C overnight each time. The eyes were frozen embedded in Tissue-Tek O.C.T embedding medium (VWR) using dry ice and 14 um sections were collected onto Superfrost PLUS slides (VWR) using a Leica CM1850 cryostat. Tissue was washed with 1X PBS for 5 minutes to remove O.C.T, followed by boiling in RNAscope Target Retrieval reagent (Advanced Cell Diagnostics, ACD) for 5 minutes. Afterwards, slides were briefly washed with sterile water and incubated for 15 minutes at 40 °C with RNAscope Protease III reagent (ACD). Fluorescent in situ hybridization staining was performed using the RNAscope Fluorescent Multiplex Detection kit (ACD) according to the user’s manual. The kcnj13 target probe and odc1 and dapB control probes were designed and provided by ACD. Slides were mounted in Prolong Gold Antifade mountant (Thermo Fisher) and imaged using a Leica LSM 710 confocal microscope.

Primary and/or metastatic liver specimens of CC were collected from seven patients who were over 18 years of age and diagnosed with stage 4 CC. All patients had moderately differentiated colorectal adenocarcinoma and underwent surgical resection or biopsy at Siriraj Hospital during the period of 2020–2021. The participants consisted of three women and four men with an average age of 64.14 years. All patients had liver-only metastasis. Seven primary tissues of stage 4 CC (five from the left colon and two from the right colon) were individually collected from all participants. Four metastatic liver tissues of stage 4 CC were individually derived from four patients who had liver metastasis and underwent surgical resection or biopsy at the metastasis site. Informed consent was obtained from all participants, and ethical approval was obtained from the Siriraj Institutional Review Board (certificates of approval no. Si 348/2019 and Si 105/2021). Formalin-fixed paraffin-embedded (FFPE) tissues were prepared, sectioned at a thickness of 5 µm, and mounted on Superfrost Plus slides (VWR International, Radnor, PA, USA) for spatial transcriptomic analysis and immunohistochemistry (IHC) staining. All samples were assessed and confirmed by a pathologist.