After fixation in 4% PFA as described above, membranes were cut from their supports with a scalpel and dehydrated through a series of baths in increasing concentrations of ethanol in water (50%, 75%, 95%, 100%, 100%). After clearing 10 min in Clearene (Leica, Germany) to replace the ethanol, samples were immersed in liquid paraffin for one hour at 60 degrees to allow complete infusion of paraffin into the cells, and then set into block moulds and allowed to solidify at RT. Sectioning was performed on a Leica RM2135 microtome, and cut sections were deposited onto slides with the aid of a heated water bath. Prior to staining or immunolabelling, paraffin was removed with 2x10 min washes in Clearene, and then immediately rehydrated with the same series of ethanol baths in reverse order. For epitope retrieval, slides were immersed in citrate buffer at 90°C for 20min.
Clearene
Manufactured by Leica
Sourced in Germany
Clearene is a laboratory equipment product designed for precise filtration and sample preparation. It features high-quality materials and advanced engineering to ensure reliable performance in scientific applications.
Automatically generated - may contain errors
Lab products found in correlation
Eclipse 80i, by Nikon (2 mentions)
Rm2135 microtome, by Leica (1 mentions)
Paraffin, by Merck Group (1 mentions)
Dig rna labeling kit, by Roche (1 mentions)
Dpx mounting media, by Merck Group (1 mentions)
Ab3508, by Abcam (1 mentions)
Microtome, by Leica (1 mentions)
Paraplast plus, by Merck Group (1 mentions)
Sc 542, by Santa Cruz Biotechnology (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions)
Fluoromount, by Southern Biotech (1 mentions)
Picrosirius red, by Abcam (1 mentions)
Autostainer xl, by Leica (1 mentions)
Histocore pearl, by Leica (1 mentions)
Rm2245 microtome, by Leica (1 mentions)
Organo mounting medium, by Merck Group (1 mentions)
Histocore arcadia c, by Leica (1 mentions)
5 protocols using clearene
1
Paraffin Embedding and Sectioning Protocol
2
In Situ RNA Hybridization of Primary Roots
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Fresh 5-d old primary roots cultured in noncompacted and compacted soils were fixed in formalin-acetic acid-alcohol solution, dehydrated using ethanol and Clearene (Leica), and then embedded in paraffin (Sigma). The antisense and sense probes were labeled using the DIG RNA Labeling Kit (SP6/T7, Roche). The procedures for in situ RNA hybridization followed the protocol described by Dreni et al. (37 (link)). Images were obtained using a Nikon microscope (Eclipse 80i). The primer sequences were listed in SI Appendix, Table S2 .
3
Black Gold II Staining for Myelin Imaging
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Frozen brain and spinal cord sections were stained with Black Gold II (BGII) (AG105, Millipore) according to manufacturer’s instructions. In brief, sections were washed in water, immersed in BGII solution at 60 °C, and incubated for 20 min, followed by incubation in 1% sodium thiosulfate at 60 °C for 5 min. Slides were rinsed in water, dehydrated, incubated in Clearene (Leica) for 2 min, and coverslipped using DPX mounting media (Sigma). Images of brain and spinal cord sections were acquired using DM5500 and Nikon Eclipse 80i microscopes.
4
Immunohistochemical Analysis of Dental Tissues
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Dental tissues were fixed by immersion in a 4% paraformaldehyde solution for 4 h. After washing in PBS, the samples were dehydrated in ethanol, rinsed in clearene (Leica-France) and paraffin-embedded (Paraplast plus, Sigma). Serial 8 μm sections were cut using a microtome (RM 2145, Leica, France). Sections were deparaffinized and rehydrated in decreasing concentrations of ethanol. Slices were microwaved for 20 min, and the tissues permeabilized with 0.5% Triton X-100 for 10 min. Sections were then washed in PBS and blocked with 10% normal goat serum in PBS for 1 h at room temperature. Slices were incubated overnight at 4°C with primary rabbit polyclonal anti-AR (N-20:sc-816, Santa Cruz) (1:200), anti-VDR (ab3508, Abcam) (1:500), or anti-ERα (sc-542, Santa Cruz) (1:50) antibodies. Sections were incubated with secondary goat anti-IgG coupled to Alexa Fluor 594 antibody (A-11072, Life Technologies) (1:500) at room temperature for one h in the dark. After rinsing with PBS, sections were immersed in DAPI (010M4003-Sigma) (1:100000) for 5 min and finally mounted with Fluoromount (Southern Biotech, Clinisciences).
5
Tissue Processing, Sectioning, and Staining
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Blood
Tissue processing sectioning and staining Tissue samples were collected and stored in 4% paraformaldehyde at 4°C overnight. Tissues were transferred into 70% (v/v) ethanol and stored at 4°C overnight. They were processed using HistoCore PEARL (Leica, Germany) tissue processor and embedded in para n with the HistoCore Arcadia C (Leica, Germany) then sectioned at 5 μm using a Leica RM2245 microtome (Leica, Germany), mounted on Polysine ® slides and dried at 40°C for at least an hour before staining. Slides were loaded into Leica Autostainer XL (Leica, Germany) and at the end of the H&E programme, glass coverslips were mounted onto the slides using Organo mounting medium (Sigma, USA) and dried at room temperature (RT). Alternatively, slides were cleared with two changes of Clearene, before hydrating successively in 100%, 95%, and 70% (v/v) ethanol gradients and deionised water. Slides were immersed in Picro-Sirius red (Abcam, United Kingdom) for 1 hour, then transferred into two changes of acetic acid followed by two changes of 100% (v/v) ethanol, cleared with two changes of Clearene (Leica, Germany) before glass coverslips were mounted onto the slides using Organo mounting medium (Sigma, USA) and dried at RT.
Tissue processing sectioning and staining Tissue samples were collected and stored in 4% paraformaldehyde at 4°C overnight. Tissues were transferred into 70% (v/v) ethanol and stored at 4°C overnight. They were processed using HistoCore PEARL (Leica, Germany) tissue processor and embedded in para n with the HistoCore Arcadia C (Leica, Germany) then sectioned at 5 μm using a Leica RM2245 microtome (Leica, Germany), mounted on Polysine ® slides and dried at 40°C for at least an hour before staining. Slides were loaded into Leica Autostainer XL (Leica, Germany) and at the end of the H&E programme, glass coverslips were mounted onto the slides using Organo mounting medium (Sigma, USA) and dried at room temperature (RT). Alternatively, slides were cleared with two changes of Clearene, before hydrating successively in 100%, 95%, and 70% (v/v) ethanol gradients and deionised water. Slides were immersed in Picro-Sirius red (Abcam, United Kingdom) for 1 hour, then transferred into two changes of acetic acid followed by two changes of 100% (v/v) ethanol, cleared with two changes of Clearene (Leica, Germany) before glass coverslips were mounted onto the slides using Organo mounting medium (Sigma, USA) and dried at RT.
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