Fetoplacental unit sections were prepared as described above and subjected to in situ hybridization with an RNAscope probe specific for the mouse gene Acer2 (Cat. # 493281) or Tpbpa (Cat. # 405511), which both were designed and manufactured by Advanced Cell Diagnostics (ACD (Newark, CA, USA). Briefly, tissue sections were baked and deparaffinized on the instrument, followed by epitope retrieval (using Leica Epitope Retrieval Buffer 2 at 95°C or at 88°C for 15 min) and protease treatment (15 min at 40°C). Probe hybridization, signal amplification, colorimetric detection, and counterstaining were subsequently performed using RNAscope® 2.5 HD reagent kits (ACD) as per the manufacturer’s instructions. The stained tissue sections were imaged under the Olympus BX53 fluorescent microscope. Brown or red dots in cells represent mRNA molecules of the genes Acer2 and Tpbpa, respectively.
Epitope retrieval buffer 2
Manufactured by Leica
Epitope Retrieval Buffer 2 is a laboratory reagent used to prepare tissue samples for immunohistochemical analysis. It is designed to facilitate the exposure of target epitopes within the sample, enabling more effective antibody binding and detection.
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Lab products found in correlation
3 protocols using epitope retrieval buffer 2
1
In situ Hybridization Analysis of Placental Genes
2
Immunohistochemical Analysis of Mammary Tumors
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Treated and control mammary tumors were fixed in 10% formalin for 48 h and then transferred to 70% ethanol until histological processing. Tumor sections were processed for Hematoxyilin–Eosin staining or immunohistochemistry using antibodies listed in Supplementary Table 1 . All the antibodies used in this study were validated by the supplier as well as independent publications from academic laboratories. However, before we use any of the antibodies, we have validated their suitability by titrating the amount of antibody and running positive and negative controls. All antibodies were stained on the Leica Bond RX Automated Stainer with either Epitope Retrieval Buffer 1 (ER1), Epitope Retrieval Buffer 2 (ER2), or with Enzyme 1 (Leica). All antibodies were diluted in Power Vision Super blocker (Leica). The IHC slides were scanned using Aperio ScanScope slide scanner (Aperio Technologies, CA), and the images were analyzed using Positivity Pixel Count 9.0 algorithm within ImageScope software (Aperio Technologies, CA) as described previously75 (link).
3
In Vivo Tracking of Collagen mRNA Expression
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Nude female mice (10-12 weeks old) were anaesthetized with isoflurane, and the dorsal skin region was injected with 50 µl 2.7 × 10 9 copy number of CNP COL1A1 mRNA EVs. At predefined timepoints (12, 24, 48, 96 h, 7 d, 10 d, 14 d) following the skin injection, the skin was excised and put in 4% formaldehyde for fixation. Subsequently, the slices were embedded in paraffin further sectioned into 4 µm slices. RNAscope automated in situ hybridization assay for detection of human COL1A1 mRNA was performed using the HybEZ II Hybridization System (Advanced Cell Diagnostics (ACD)), and all in situ hybridization reagents were ACD products. In brief, target retrieval was performed at 95 °C for 15 min using Leica Epitope Retrieval Buffer 2, followed by protease treatment at 42 °C for 15 min. The probe (RNAscope Probe-Hs-COL1A1, 401891, ACD) was hybridized for 2 h at 40 °C followed by RNA scope amplification, and RNAscope 2.5 HD Assay BROWN kit was used for visualization of staining. RNAscope 2.5 LS probe-Rn-Ppib was used as negative control.
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