Livers were rinsed with PBS and fixed with 4% PFA in PEM buffer (100 mM PIPES pH 6.9, 5 mM MgCl2, 5 mM EGTA). After fixation, the fixative buffer was replaced by 15–30% sucrose series, embedded in Tissue-Tek optimal cutting temperature (Sakura Finetek) and stored at −80 °C. OCT-embedded livers were cut to a thickness of 10 μm. Whole-blood samples were fixed in PEM buffer with acid citrate-dextrose. Liver sections were stained with antibodies against tubulin, HNF4 and F4/80. Blood cells were stained with antibodies against tubulin. Nuclei were labelled with DAPI. Images were acquired on a Zeiss Axio Observer Z1 equipped with a Plan Apo ×63/1.4 numerical aperture DIC objective and an LSM780 single-point scanning confocal scan head. Images were collected using ZEN black SP5.
Tissue tek optimal cutting temperature
Manufactured by Sakura Finetek
Sourced in United States
The Tissue-Tek optimal cutting temperature (O.C.T.) compound is a fast-freezing embedding medium designed for cryosectioning of tissue samples. It is formulated to provide a matrix that supports and protects the tissue during the freezing process, enabling thin, uniform sections to be cut on a cryostat.
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4 protocols using tissue tek optimal cutting temperature
1
Immunohistochemical Characterization of Liver Tissue
2
Tissue Fixation and Sectioning
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Intestinal jejunal tissue and distal colon and pancreatic tissue were fixed in 10% neutral buffered formalin (Thermo Fisher Scientific, Waltham, MA) containing zinc (for paraffin) or 4% paraformaldehyde (for frozen sections) overnight at 4°C and embedded in paraffin or Tissue-Tek Optimal Cutting Temperature (Sakura Finetek USA, Torrance, CA), respectively. Tissue for frozen sectioning was cryoprotected in a 30% sucrose solution (Macron, Center Valley, PA) for an additional night at 4°C; 10-μm sections were utilized.
3
Cryosectioning TA Muscle Samples
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A Leica CM1850 cryostat was used with Tissue-TEK optimal cutting temperature compound-embedded TA muscles (Sakura Finetek USA) as described previously.11 (link)
4
Immunocytochemistry of adrenal, ovary, and testis
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Immunocytochemistry was performed as previously described (2 (link)). Briefly, adrenal, ovary, and testis tissues were fixed in 10% formalin overnight before processed and embedded in paraffin for section. Slides were sectioned at 10 μm and stained with anti-Bscl2 (1:200 dilution; Thermo Fisher Scientific, Waltham, MA) or mouse IgG as negative control. For Oil Red O staining, adrenals were fixed in Tissue-Tek optimal cutting temperature (Sakura Finetek USA, Torrance, CA) and processed into 10 μm sections before staining with Oil Red O. Images were quantified using ImageJ software from the National Institutes of Health.
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