Tongues were snap-frozen in dry ice-cooled 2-methylbutane (Acros, Geel, Belgium), and embedded in cryomatrix compound (Thermo Fisher Scientific, Waltham, MA). A Leica CM 3050S (Leica Microsystems, GmbH, Nussloch, Germany) cryostat with installed CryoJane®, was used for cryosectioning. Frozen 7 μm tissue sections were mounted onto commercial CryoJane® glass slides. An Arcturus® laser capture microscope (Thermo Fisher Scientific) was used to retrieve epithelial tissue from tongue dorsal and ventral surfaces. Samples were pooled and solubilized in Cell Lysate Buffer® (Signosis, Sunnyvale, CA) for direct reverse transcription, and relative cDNA levels were quantified by RT-qPCR, as described above.
Cryojane
Manufactured by Leica
Sourced in Germany, Japan, United States
Cryojane is a cryogenic microtome system designed for the ultra-thin sectioning of frozen biological samples. The core function of Cryojane is to provide precise and controlled sectioning of samples maintained at cryogenic temperatures, enabling detailed examination and analysis of their structural and morphological characteristics.
Automatically generated - may contain errors
Lab products found in correlation
Cryostat, by Leica (5 mentions)
Antifade mounting medium with dapi, by Vector Laboratories (3 mentions)
Alexa fluor 647 goat anti rabbit, by Thermo Fisher Scientific (2 mentions)
Ab22552, by Abcam (2 mentions)
Alexa fluor 488 goat anti rabbit, by Thermo Fisher Scientific (2 mentions)
Confocal microscope, by Leica (2 mentions)
Nb600 600, by Novus Biologicals (2 mentions)
Ab5535, by Merck Group (2 mentions)
Alexa fluor 488 goat anti rabbit igg, by Thermo Fisher Scientific (2 mentions)
Cryomatrix compound, by Thermo Fisher Scientific (1 mentions)
2 methylbutane, by Thermo Fisher Scientific (1 mentions)
Cm3050, by Leica (1 mentions)
Power block, by BioGenex (1 mentions)
Goat anti gfp fitc, by Abcam (1 mentions)
Alexa fluor 488 anti rabbit, by Thermo Fisher Scientific (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Vector Laboratories (1 mentions)
Nuclear fast red, by Vector Laboratories (1 mentions)
Anti pcna sc 56, by Santa Cruz Biotechnology (1 mentions)
Alexa fluor 594, by Thermo Fisher Scientific (1 mentions)
C1 confocal system, by Nikon (1 mentions)
12 protocols using cryojane
1
Cryo-Sectioning and Laser Capture Microdissection of Tongue Tissues
2
Cryosectioning and Immunofluorescent Staining
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Frozen samples were sectioned at 10 μm using CryoJane (Leica Microsystems). The slides were blocked in 1× Power Block (BioGenex) in PBS at room temperature for 1 h, then stained with primary antibody diluted in blocking buffer overnight at 4 °C. The next day, the slides were washed in PBS 3 × 5 min, incubated in secondary antibody in blocking buffer for 1 h at room temperature, and washed in PBS 3 × 5 min before mounting with DAPI (Vectashield). The primary antibodies used were goat anti-GFP (FITC) (1:200; abcam) and rabbit anti-beta galactosidase (abcam). Anti-Rabbit-AlexaFluor488 (Life Technologies) was used for the secondary antibody for immunofluorescent staining.
3
Comprehensive Tissue Histology Protocol
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PT tissue was harvested as described for qPCR and embedded in OCT with the orientation noted. Samples were flash frozen on a liquid nitrogen-cooled metal block and kept at -80°C until required.
Frozen tissue was sectioned on a cryostat at -22°C at 10 µm thicknesses in either the frontal or sagittal plane. Serial sections were acquired from the mid-region of each sample and recovered onto adhesive coated slides (CryoJane; Leica Microsystems Inc.). H&E staining was used to assess the general structure, Oil Red-O staining for evaluating tissue lipid content, and IHC for examining macrophage phenotypes. Perls Prussian blue was used as a global stain for iron-containing macrophages and counter-stained with nuclear fast red or Safranin-O. Toludine blue was used to visualize mast cells.
T A B L E 1 Oligonucleotide sequence of primers of selected rat genes.
Frozen tissue was sectioned on a cryostat at -22°C at 10 µm thicknesses in either the frontal or sagittal plane. Serial sections were acquired from the mid-region of each sample and recovered onto adhesive coated slides (CryoJane; Leica Microsystems Inc.). H&E staining was used to assess the general structure, Oil Red-O staining for evaluating tissue lipid content, and IHC for examining macrophage phenotypes. Perls Prussian blue was used as a global stain for iron-containing macrophages and counter-stained with nuclear fast red or Safranin-O. Toludine blue was used to visualize mast cells.
T A B L E 1 Oligonucleotide sequence of primers of selected rat genes.
4
Spinal Cord Tissue Processing and Staining
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For embryos and early postnatal mice, the spinal column containing the spinal cord was dissected and fixed in 1% (wt/vol) paraformaldehyde in PBS at 4 °C overnight. The tissue was then washed in PBS, incubated in 30% sucrose solution in PBS for 48 h at 4 °C, and embedded in OCT. The spinal cord of adult mice was fixed through intracardial perfusion for 10 min at room temperature with 4% (wt/vol) paraformaldehyde in PBS. The spinal column containing spinal cord was then postfixed in 1% (wt/vol) paraformaldehyde in PBS at 4C overnight. Spinal cord was dissected from the spinal column, washed in PBS, incubated in 30% sucrose solution in PBS for 48 h at 4 °C, and embedded in OCT.
Frozen samples were sectioned at 12 μm using CryoJane (Leica Microsystems) and postfixed with 0.1% (wt/vol) paraformaldehyde on ice, washed in detergent rinse (PBS with 2 mM MgCl2 0.01% sodium deoxycholate, and 0.02% Nonidet P-40) and stained in staining solution (PBS with 2 mM MgCl2, 0.01% sodium deoxycholate, 0.02% Nonidet P-40, 5 mM potassium ferricyanide, 5 mM potassium ferrocyanide, and 1 mg/mL X-gal) in a dark room at room temperature overnight. Sections were then washed and counterstained with Nuclear Fast Red (Vector Laboratories), followed by dehydration sequentially with an ethanol gradient (50%, 70%, 90%, and 100%).
Frozen samples were sectioned at 12 μm using CryoJane (Leica Microsystems) and postfixed with 0.1% (wt/vol) paraformaldehyde on ice, washed in detergent rinse (PBS with 2 mM MgCl2 0.01% sodium deoxycholate, and 0.02% Nonidet P-40) and stained in staining solution (PBS with 2 mM MgCl2, 0.01% sodium deoxycholate, 0.02% Nonidet P-40, 5 mM potassium ferricyanide, 5 mM potassium ferrocyanide, and 1 mg/mL X-gal) in a dark room at room temperature overnight. Sections were then washed and counterstained with Nuclear Fast Red (Vector Laboratories), followed by dehydration sequentially with an ethanol gradient (50%, 70%, 90%, and 100%).
5
Cryosectioning and Immunohistochemistry Protocol
6
Quantifying TM Cell Proliferation in ROCKi-Treated Murine Eyes
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To investigate the proliferation of TM cells in ROCKi-treated tissue, we assessed the effect of Y-27632 on Tg-MYOCY437H mice. Murine eyes were embedded in Optimal Cutting Temperature Compound (Sakura; Tokyo, Japan), frozen, and sectioned to 8-μm thickness on a cryostat equipped with a tape transfer system (CryoJane, Leica). Samples were fixed in 95% ETH for 5 min and rinsed in DPBS for 5 min. Triton X-100 (0.3%) in DPBS was added to increase the permeability for 5 min; samples were then washed twice with DPBS. Samples were incubated with phalloidin (1:1000) in the blocking solution for 1 h at RT. Samples were then washed three times and incubated with DAPI for 20 min at RT. After an additional wash with DPBS, coverslips were mounted on the samples by using Neutral Balsam (Solarbio), and images were obtained using confocal microscopy.
7
Mouse Tibia Fixation and Sectioning
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Two-month-old mice were perfused with 4% paraformaldehyde (PFA) as described previously.21 (link) After perfusion, tibias were dissected and fixed in 4% PFA at 4 °C overnight. The fixed tibias were decalcified in 14% EDTA (pH 7.4) for 3 days, incubated in 30% sucrose at 4 °C overnight and then snap-frozen in optimal cutting temperature (OCT) embedding medium. Frozen sections were cut at 8 μm thickness with a cryostat equipped with CryoJane (Leica, Buffalo Grove, IL, USA). The sections were kept at −20 °C until analyses.
8
Assessing Embryonic Limb Development
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Pregnant female mice were injected intraperitoneally with EdU (Thermo Fisher Scientific, C10339) at 10 µg/g body weight 2 h before harvest. Embryonic limbs were dissected out, fixed with 4% paraformaldehyde (EMS, 15710) overnight, decalcified for 3 days with daily changes of 14% EDTA (pH 7.2), and incubated in 30% sucrose overnight for cryoprotection prior to embedding in optimal cutting temperature (OCT) (Tissue-Tek, 4583). Sections of 10 μm in thickness were obtained with a Leica cryostat equipped with Cryojane (Leica). EdU was detected by a Click reaction performed according to the manufacturer’s instructions. TUNEL assay was performed with the In-Situ Cell Death Detection Kit Fluorescein (Roche, 11684795910). For immunostaining, the frozen sections were stained with specific antibodies against Glut1 (Santa Cruz, SC-7903), Endomucin (Santa Cruz, SC-65495), Osterix (Abcam, ab22552), Mmp13 (Abcam, ab39012), or Collagen type X (DSHB Hybridoma Product, X-AC9). The secondary antibodies were as follows: Alexa Fluor Alexa Fluor 488 goat anti-rabbit IgG (Life Science, A-11034), Alexa Fluor 594 goat anti-rabbit IgG (Life Science, R37117). Stained section were mounted with VECTASHIELD mounting medium containing DAPI (Vector Laboratories, H-1200).
9
Immunofluorescence Analysis of Bone Markers
10
Histological Analysis of Mandibular Bone Cells
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Mandibles intended for histological staining were embedded in paraffin using standard histological procedures, then sectioned at 5-μm thickness for Masson's trichrome, Sirius red, and TRAP staining as previously reported (Wang et al., 2017 (link)). Samples for cell lineage tracing were dehydrated with 30% sucrose and embedded in OCT. Next, 10-μm-thick sections were prepared with a Leica cryostat equipped with Cryojane as previously reported (Xie et al., 2019 (link)). Immunostaining was then carried out as previously described (Wang et al., 2020 (link)) using the following primary antibodies: anti-OSX rabbit antibody (1:200, ab22552), anti-PERIOSTIN goat antibody (1:400, AF2955), anti-MEPE rabbit antibody (1:100, LF-155), anti-SOST goat antibody (1:100, AF1589), anti-PCNA rabbit antibody (1:100, Cst13110s). The secondary antibodies used for immunostaining: Goat anti-Rabbit IgG-Alexa Fluor 488 (1:200, Invitrogen); Rabbit anti-Goat IgG-Alexa Fluor 488 (1:200, Invitrogen); and Goat Anti-Rabbit IgG-unconjugated (1:100, Vector laboratories).
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