For immunohistochemical examinations of the small intestine, pancreas, and lung, the mice were perfused with 1% paraformaldehyde (PFA) (Sigma-Aldrich), and the isolated tissues were fixed with 4% PFA in PBS for 24 hr and embedded in FSC 22 Clear Frozen Section Compound (Leica Biosystems). Then, 10-μm-thick frozen sections were cut on a cryostat. The sections were blocked with Block-Ace (DS Pharma Biomedical) and 0.1% Triton X-100 in PBS. Primary or secondary antibodies were incubated for 2 hr or 1 hr, respectively, at ambient temperature. All primary antibodies were used at 1:1,000, and all secondary antibodies were at 1:500. For immunofluorescence of cultured cells, MDCK-pTR GFP-RasV12 cells were mixed with MDCK cells at a ratio of 1:50 and cultured on the collagen matrix as previously described (Hogan et al., 2009 (link)). The mixture of cells was incubated for 8 hr until they formed a monolayer, followed by tetracycline treatment for 24 hr. Cells were fixed with 4% PFA in PBS and permeabilized with 0.5% Triton X-100 in PBS, followed by blocking with 1% BSA in PBS. Alexa-Fluor-568-conjugated phalloidin was incubated for 1 hr at ambient temperature. Immunofluorescence images of mouse tissues and cultured cells were acquired using the Olympus FV1000 system and Olympus FV10-ASW software. Images of TMRM were quantified with the MetaMorph software (Molecular Devices).
Fsc 22 clear frozen section compound
Manufactured by Leica
Sourced in Germany
The FSC 22 Clear Frozen Section Compound is a laboratory product designed for the preparation of frozen tissue sections. It is a clear, water-soluble medium that provides support and stabilization for tissue samples during the freezing process, ensuring the preservation of the tissue's structural integrity.
Automatically generated - may contain errors
Lab products found in correlation
Block ace, by Sumitomo Pharma (2 mentions)
Metamorph software, by Molecular Devices (2 mentions)
Fv10 asw software, by Olympus (2 mentions)
Fv1000 system, by Olympus (2 mentions)
Cellsens 1, by Olympus (1 mentions)
Dp80 microscope, by Olympus (1 mentions)
Rnalater, by Qiagen (1 mentions)
Mildfolm, by Fujifilm (1 mentions)
Cm1900, by Leica (1 mentions)
Anti calbindin d 28k, by Merck Group (1 mentions)
X tra adhesive glass slides, by Leica (1 mentions)
Cryostar nx70, by Thermo Fisher Scientific (1 mentions)
Anti mouse igg2a, by Thermo Fisher Scientific (1 mentions)
Ab5694, by R&D Systems (1 mentions)
A1r confocal microscope, by Nikon (1 mentions)
Mab1195, by R&D Systems (1 mentions)
Fluoromount, by Diagnostic BioSystems (1 mentions)
Cm1520 cryostat, by Leica (1 mentions)
Permanent mounting medium, by Thermo Fisher Scientific (1 mentions)
Eclipse ts2, by Nikon (1 mentions)
7 protocols using fsc 22 clear frozen section compound
1
Immunohistochemical Analysis of Mouse Tissues
2
Hematoxylin and Eosin Staining of Skin Lesions
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Hematoxylin and eosin (H&E) staining was performed as previously described [41 (link)]. Briefly, dorsal skin lesions were obtained from the control (PBS), minoxidil (5%), and EF-2001 (0, 5, and 50 mg/mL) groups, fixed in 10% formalin, and stabilized in 10, 15, and 20% sucrose solution. To embed the fixed skin in a cryomold, the FSC 22 Clear frozen section compound (Cat. no. 3801480; Leica Biosystems, Wetzlar, Germany) was used. Afterward, the cryo-tissues were sectioned, stained using H&E, visualized under an Olympus DP80 microscope, and analyzed using imaging software (Cell Sens 1.8; Olympus Europa SE & Co., KG, Hamburg, Germany).
3
Liver Transplantation Monitoring Protocol
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Body weight (BW) measurement and blood sample collection were performed at the time of GCV administration, and weekly after PH transplantation. Sera were stored at - 20 °C until use, and they were subjected to the measurement of serum ALT levels (SRL Inc. Aichi, Japan). In all the models (Sham, Auto, Allo, FK, and ASC models), the mice were sacrificed at before (day 0) and 1 week after GCV administration (0 w), 1, 2, 4, and 8–11 weeks after PH transplantation (1, 2, 4, and 8 w, respectively). The resected livers were subjected to the histological analysis, flow cytometric analysis for hepatocyte polyploidy, and hepatic gene expression analysis (Fig. 1 b and c). The part of liver tissues was fixed with 10 N Mildfolm (Wako) at room temperature for the histological examination. The unfixed liver tissues were embedded with FSC 22 Clear Frozen Section Compound (Leica Biosystems, Germany), and frozen in liquid nitrogen for the preparation of frozen specimens also for another histological examination. The remaining part of them were stored at -30 °C in RNAlater (QIAGEN, Hilden, Germany) for the analysis of mRNA expression. For the flow cytometric analysis, the livers were perfused as described above (2.2 PH isolation) to collect isolated hepatocytes.
4
Calbindin Expression in NPC1 Mice
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10-weeks-old wt and Npc1nmf164 mice either sham-, ORX-301-, or HPβCD-treated were anaesthetized by intraperitoneal injection of a mixture of xylazine/ketamine (20–34 mg/kg) and then transcardially perfused with PBS. Brains were quickly dissected, fixed overnight at 4 °C in 4% PFA and processed as previously described52 (link). Briefly, fixed brains were dehydrated by incubation in 15–30% sucrose in PBS, embedded in FSC 22 Clear Frozen Section Compound (Leica Biosystems, Milano, Italy) and serially sectioned (slice thickness 10 µm) using a cryostat (Leica CM 1900). Sagittal sections were mounted on X-tra Adhesive glass slides (Leica biosystems). Sections were processed for immunofluorescence using a mouse monoclonal Calbindin antibody (Anti-Calbindin D28K, Sigma, Milan, Italy; 1:500 dilution in PBS) as routinely performed in our lab. A permeabilization step performed by incubating sections in 0.1% Triton X-100 in PBS for 10 min was followed by a 3 h incubation in a blocking solution containing 5% goat serum, 1% BSA, 0.2% Triton X-100 in PBS. Sections were then incubated over night with a 1:500 primary antibody dilution in PBS and, after several washes with PBS, with a TRITC-conjugated anti-mouse secondary antibody (Zymed-Thermo Fisher Scientific, Milan, Italy).
5
Immunohistochemical Analysis of Mouse Tissues
6
Immunohistochemical Analysis of Chicken Hindgut
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A hindgut was dissected from chicken embryo and fixed in 4% (w/v) paraformaldehyde (PFA)/phosphate buffered saline (PBS: 0.14 M NaCl, 2.7 mM KCl, 10 mM Na2HPO4–12H2O, 1.8 mM KH2PO4) for 10 min at room temperature (RT). The specimen was washed in PBS twice for 5 min each at RT, and embedded in FSC 22 Clear frozen section compound (Leica, 3801480). Cryostat sections of 20 μm were prepared (Thermo Scientific, Cryostar NX70). Following drying on the hotplate at 37°C, the sections were re-fixed in 4% PFA for 5 min at RT. After washing three times in PBS for 5 min each at RT, the sections were incubated with 0.5% blocking reagent (Roche, 1096176)/PBS for 1 h at RT, followed by primary antibodies; 1:300 dilution of anti-αSMA (abcam, ab5694) and 1:300 dilution of Tuj1 (R & D systems, MAB1195) overnight at 4°C. Following three times washing in PBS for 5 min each at RT, they were incubated with Alexa 488-conjugated second antibodies; 1:300 dilution of anti-rabbit IgG (H + L) (donkey; Invitrogen, A21206), 1:300 dilution of anti-mouse IgG2a (goat; Invitrogen, A21131) and 1:2000 dilution of DAPI (Nacalai Tesque, 11034–56) for 1.5 h at RT. After washing three times for 10 min each at RT, the specimens were sealed with Fluoromount (Diagnostic BioSystems). Fluorescent images were obtained using a Nikon A1R confocal microscope.
7
Cresyl Violet Staining of Mouse Brain Sections
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The mice were euthanized in a CO2 chamber at 33 days after rhEPO administration. The mice were perfused with 30 mL of 1× PBS. The extracted brains were fixed in 4% paraformaldehyde for 24 h and then dehydrated in 30% sucrose solution. Brain samples were frozen with FSC 22 Clear Frozen Section Compound (Leica #3801480, Wetzlar, Germany) and 20 μm-thick coronal slices cut using a Leica CM1520 cryostat. Mounted frozen sections of positively charged slides were air-dried overnight and then placed in distilled water for washing. After being stained with 0.1% Cresyl violet solution prepared with Cresyl violet acetate (Sigma-Aldrich #C5042, St. Louis, MO, USA) for 4 min, the slides were rinsed with 1× PBS for 1 min. Then, they were dehydrated in 70%, 80%, 90%, and 95% ethanol solutions for 2 min each. They were cleared in xylene solution for 5 min and immediately mounted with the Permanent™ mounting medium (Fisher Chemical™ #SP15, Hampton, VA, USA). Photographs of the prepared slides were taken with the i-solution™ Auto Plus (IMT, Vancouver, BC, Canada) program using an ECLIPSE Ts2 (Nikon, Tokyo, Japan) instrument. The photos were taken at magnifications of 100× and 400×. Cresyl violet acetate solution stained the Nissl material in the cytoplasm of neurons, and the neuropil was stained with granular purple-blue. This stain was used to identify neural structures.
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