Frozen sections (7µm) were obtained using the CryoJane system (Leica Biosystems, Nussloch, Germany). RNAscope assay was conducted with RNAscope BROWN kit according to the manufacturer’s instructions (Advanced Cell Diagnostics, Hayward, CA). The negative and positive controls were performed on the same samples using the negative and positive probes from the kit. Briefly, the endogenous peroxidase was blocked with the provided pretreatment 1 solution for 10 min at RT, followed by epitope retrieval by boiling in the provided buffer for 5 min, protease digestion for 30 min at 40°C in a hybridization oven (HybEZ Oven, Advanced Cell Diagnostics, Hayward, CA) and target hybridization for 2 h at 40°C followed by six sequential amplification steps. Sections were counterstained with Hematoxylin. Brown punctuate dots in the section are considered positive staining.
Cryojane system
Manufactured by Leica
Sourced in Germany
The CryoJane system is a cryostat designed for sectioning frozen tissue samples. It provides consistent and reliable sectioning of frozen samples for various microscopy and analysis applications.
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Lab products found in correlation
2 protocols using cryojane system
1
RNAscope Assay on Frozen Sections
2
Immunohistochemical Analysis of Mouse Femur
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Mouse femurs were fixed in 10% formalin for 24 to 48 hours at 4°C. Bones were then decalcified in 14% EDTA, pH 7.4, for 10 to 14 days at 4°C. Following incubation in 30% sucrose for 24 hours at 4°C to dehydrate, bones were embedded in optimal cutting temperature compound (OTC, Sakura Finetek). These tissue blocks were cut into 12-μm sections using a CryoJane system (Leica Biosystems). For immunostaining, the slides were blocked with 5% donkey serum in TBST for 1 hour at room temperature. Slides were then incubated with primary antibody overnight at 4°C, followed by incubation with secondary antibody for 1 hour at room temperature. The following antibodies were used: rabbit anti–collagen I (Abcam, ab34710), rabbit anti–collagen III (Abcam, ab7778), goat anti–TGF-β1 (LAP) (R&D Systems, AB-246-NA), PE–anti-CD41 (BD Biosciences, MWReg30), and Alexa Fluor 647–conjugated donkey anti–rabbit IgG (H+L) (Thermo Fisher Scientific). Finally, slides were mounted with ProLong Gold antifade reagent with DAPI (Life Technologies). Images were acquired with an LSM 700 microscope (Carl Zeiss). Fluorescence intensity was quantified using ImageJ (NIH).
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