Tissues were formalin-fixed and paraffin-embedded. RNA-ISH was performed using the RNAscope protocol from Advanced Cell Diagnostics, Inc. (Hayward, CA, USA). Paraffin sections were deparaffinized, rehydrated in graded ethanol, and then H2O2 was applied. Sections were processed as previously described [27 (link)]. A RNAscope 2.5 HD Reagent kit-RED was used for chromogenic labeling. Tissues were mounted using ProLong Gold Antifade Mountant with DAPI (Invitrogen, Waltham, MA, USA) [27 (link)]. Counts for p16Ink4a- and p21Waf1/Cip1-positive cells were calculated using Fiji-ImageJ software.
Rnascope protocol
Manufactured by Advanced Cell Diagnostics
Sourced in United States
The RNAscope protocol is a technique used for the detection and localization of specific RNA molecules in tissue samples. It utilizes advanced in situ hybridization technology to visualize and analyze the expression of target genes at the cellular level.
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9 protocols using rnascope protocol
1
RNA-ISH for p16Ink4a and p21Waf1/Cip1 expression
2
RNA-ISH for p16 and eGFP Detection
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RNA‐ISH was performed after RNAscope protocol from Advanced Cell Diagnostics, Inc. (ACD). Paraffin sections were deparaffinised with Histoclear, rehydrated in graded ethanol (EtOH), and H2O2 was applied for 10 min at RT followed by two washes in H2O. Sections were placed in hot retrieval reagent and heated for 30 min. After washes in H2O and 100% EtOH, sections were air‐dried. Sections were treated with protease plus for 30 min at 40°C, washed with H2O and incubated with target probe (p16, eGFP) for 2 h at 40°C. Afterwards, slides were washed with H2O followed by incubation with AMP1 (30 min at 40°C) and next washed with wash buffer (WB) and AMP2 (15 min at 40°C), WB and AMP3 (30 min at 40°C), WB and AMP4 (15 min at 40°C), WB and AMP5 (30 min at RT) and WB and, finally, AMP6 (15 min at RT). Finally, RNAscope 2.5 HD Reagent kit‐RED was used for chromogenic labelling. After counterstaining with haematoxylin, sections were mounted and coverslipped. All analysis was performed in a blinded manner.
3
Multiplexed RNA Detection in Tissue Sections
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RNA‐ISH was performed per the RNAScope protocol from Advanced Cell Diagnostics Inc. (ACD): RNAScope Multiplex Fluorescent Assay v2. Briefly, the assay allows simultaneous visualization of up to 4 RNA targets, with each probe assigned to a different channel (C1, C2, or C3 or C4). p21 (CDKN1A) signal amplified using the HRP‐C1 linked with a secondary fluorophore, Opal 570 (detected in the Cy3 range), followed by a subsequent detection of p16/p19 (CDKN2A) –c3 or EGFP‐c2 probe, the signal is amplified by the HRP‐C3 or HRP‐C2 (respectively) linked with a secondary fluorophore, Opal 650 (detected in the Cy5 range). Tissue sections were then mounted using ProLong Gold Antifade Mountant with DAPI (Invitrogen). Sections were imaged using in‐depth Z‐stacking. RNA‐ISH‐positive cells on the bone surface were quantified normalized against total cells on the bone surface.
4
RNAscope-based RNA In Situ Hybridization
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RNA-ISH was performed after RNAscope protocol from Advanced Cell Diagnostics Inc. (ACD). Paraffin sections were deparaffinized with Histoclear, rehydrated in graded ethanol (EtOH) and H2O2 was applied for 10 min at RT followed by two washes in H2O. Sections were placed in hot retrieval reagent and heated for 30 min. After washes in H2O and 100% EtOH, sections were air dried. Sections were treated with protease plus for 30 min at 40 °C, washed with H2O and incubated with target probe (p16, eGFP) for 2 h at 40 °C. Afterwards, slides were washed with H2O followed by incubation with AMP1 (30 min at 40 °C) and next washed with wash buffer (WB) and AMP2 (15 min at 40 °C), WB and AMP3 (30 min at 40 °C), WB and AMP4 (15 min at 40 °C), WB and AMP5 (30 min at RT) and WB and, finally, AMP6 (15 min at RT). Finally, RNAscope 2.5 HD Reagent kit-RED was used for chromogenic labelling. After counterstaining with haematoxylin, sections were mounted and coverslipped.
5
Spatial Transcriptomics of Developing Mouse Brain
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Embryonic samples at E12 and E14, prepared as outlined above, were submitted to the RNAscope protocol (Advanced Cell Diagnostics), following the manufacturer’s instructions with minor modifications. All RNAscope probes (as listed in the Key resources table) were purchased from ACD. Additional in situ images were obtained from the Allen Developing Mouse Brain Atlas (Figure 1G and H , Figure 1—figure supplement 2 ) and from the Allen Mouse Brain Atlas (Lein et al., 2007 (link); Figure 1—figure supplement 3B and C ).
6
Lgr5 and Hopx Expression Mapping
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RNA in situ probe hybridization and detection was performed using RNAScope protocol according to manufacturer’s instructions (Advanced Cell Diagnostics/Bio-Techne). The probes used were RNAscope Probe- Mm-Lgr5 ACDBio Cat# 312171 and RNAscope Probe- Mm-Hopx-C2 ACDBio Cat# 405161-C2. RNAScope slides were imaged and quantified by counting visible RNA positive puncta and averaging puncta per crypt or puncta per 5000μm2 in lesions.
7
RNA In Situ Hybridization Protocol
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RNA in situ hybridization was performed after RNAscope protocol from Advanced Cell Diagnostics. Paraffin sections were deparaffinized with Histoclear, rehydrated in graded ethanol, and H2O2 was applied for 10 min at room temperature, followed by two washes in H2O. Sections were placed in hot retrieval reagent and heated for 15 min. After washes in H2O and 100% ethanol, sections were air dried. Sections were treated with protease plus for 30 min at 40°C, washed with H2O, and incubated with target probe (p21, no. 408551) for 2 h at 40°C. Afterward, slides were washed with H2O, followed by incubation with AMP1 (30 min at 40°C) and next washed with wash buffer (WB) and AMP2 (15 min at 40°C), WB and AMP3 (30 min at 40°C), WB and AMP4 (15 min at 40°C), WB and AMP5 (30 min at room temperature) and WB, and finally, AMP6 (15 min at room temperature). Finally, an RNAscope 2.5 HD Reagent kit-RED was used for chromogenic labeling. After counterstaining with hematoxylin, sections were mounted.
8
Mapping Neuronal Activation Patterns
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Ninety minutes from the start of the CPP test [i.e., when Fos protein expression is known to reach peak (23 (link))], rats were deeply anesthetized and transcardially perfused with 4% paraformaldehyde for immunohistochemistry or decapitated before harvesting fresh brains to flash freeze for RNAscope in situ hybridization experiments. Brains were processed according to standard immunohistochemical procedures as previously described (24 (link)). A rabbit monoclonal c-Fos antibody (catalog number 2250; Cell Signaling Technology) was used at a dilution of 1:8000 and DAB to visualize Fos-expressing activated neurons. RNAscope protocols (Advanced Cell Diagnostics) were used to prepare flash-frozen tissue for in situ hybridization using custom-made antisense messenger RNA (mRNA) probes. The mRNA probes c-Fos (activated neurons), Slc17a7 (glutamatergic neurons), Slc32a1 (GABAergic [gamma-aminobutyric acidergic] and glycinergic neurons), and Chat (cholinergic neurons) were used to identify the specific phenotype of activated neurons.
9
Mapping Neuronal Activation Patterns
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