Organoids were fixed for 45 min in 10% neutral buffered formalin (HT501128, Sigma) and washed in 1× PBS. Organoids were placed in 25% sucrose in 0.1 M phosphate buffer solution overnight, and then mounted in Tissue-Tek O.C.T. compound (4583, Sakura), placed on dry ice to freeze, and stored at −80°C. Organoids were cryosectioned in 10 μm sections. Slides were air dried for 6 h to overnight, then postfixated for 15 min in 10% neutral buffered formalin (HT501128, Sigma) and washed in 1× PBS. Slides were dried and stored at −80°C and used within 3 months.
Ht501128
Manufactured by Merck Group
Sourced in United States, Germany
The HT501128 is a laboratory equipment product from Merck Group. It is a precision instrument designed for scientific research and analysis. The core function of this product is to facilitate accurate and reliable measurements and data collection in a laboratory setting. Further details about the intended use or specific applications of this product are not available.
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155 protocols using ht501128
1
Cryopreservation of Organoid Samples
2
Fetal Retina and Eye Cryosectioning
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Day 122 fetal retina (sex and left/right eye information unknown) and day 130 fetal eye (sex and left/right eye information unknown) were enucleated and flash frozen on dry ice and stored at −80°C. The fetal eye was allowed to come to RT in 1× PBS. The anterior portion of the eye (lens, cornea, and iris) was removed with scissors. The posterior portion of the eye was fixed overnight in 10% neutral buffered formalin (HT501128, Sigma) and washed in 1× PBS. The whole posterior eye was mounted in Tissue-Tek O.C.T. compound (4583, Sakura), placed on dry ice to freeze, and stored at −80°C. The eye was cryosectioned in 20 μm sections starting from the anterior side to the posterior, such that each section contains the nasal/temporal and dorsal/ventral information at each position along the anterior/posterior axis. For the day 130 fetal eye, sections were collected at 100 μm intervals, moving in a temporal to nasal direction. Sections were postfixed for 15 min in 10% neutral buffered formalin (HT501128, Sigma) and washed in 1× PBS, then air dried overnight. Slides were stored at −80°C and used within 6 months.
3
Histological Analysis of Murine Organs
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Mice were euthanized with isoflurane followed by cervical dislocation. Organs were manually dissected, washed in PBS, fixed overnight in 10% neutral buffered formalin (HT501128, Sigma-Aldrich), and preserved in 70% ethanol. For histology, tissue was embedded in paraffin and 5 mm sections were cut using a microtome. Hematoxylin and eosin (H&E) tissue staining was performed using HAE-2-IFU (ScyTek Laboratories, Inc). Briefly, slides were deparaffinized in xylene, followed by sequential incubations in 100%, 95%, and 70% ethanol. Tissue sections were stained with Mayer’s Hematoxylin (Lillie’s Modification) for 5 min, washed with two changes of distilled water, incubated with Bluing Reagent for 10–15 s, washed with two changes of distilled water, washed with ethanol, stained with Eosin Y Solution (Modified Alcoholic) for 2–3 min, and washed with three changes of ethanol. Some of the histology work was performed as a fee-for-service by Dr. Jonathan Fox (Department of Veterinary Sciences, University of Wyoming), including H&E, S100, and Melan A staining.
4
Histological Analysis of Mouse Livers
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5
Alveolar Bone Biopsy Protocol
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Briefly, a mucoperiosteal flap was raised to expose the alveolar ridge. No releasing incisions were performed to preserve the periosteum and ensure adequate vascularization and venous drainage in the operative zone. Initially, a core biopsy of 1.7 mm internal diameter and a depth of 8–10 mm was performed from the alveolar site to obtain bone samples from the implant beds using a trephine bur (Inside shaft ø1.7 mm, 2.35 mm × 30 mm length; 7 teeth graduation, Helmut Zepf, Seitingen-Oberflacht, Germany) (Figure 2 a–f). The obtained bone samples were divided into two pieces where one piece was stored at −80 °C for RNA isolation and the other piece was fixed in 10% neutral buffered formalin (HT501128, Sigma-Aldrich, Taufkirchen, Germany) for histological investigations.
6
Morphology of hADSC Spheres on Ca-Alginate
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The morphology of hADSC spheres on Ca-Alginate scaffolds was observed by a SEM (TM-1000, Hitachi, Tokyo, Japan). The sample was fixed with 4% formaldehyde (HT501128, Sigma, USA) for 2 h and dehydrated using a graded ethanol series (30–100%). All the samples were dried by critical point drying method and sputter-coated with platinum.
7
Histological Analysis of Mouse Testis
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Testis and caudal epididymis and other tissues were dissected immediately following euthanasia of mice. Tissues were then fixed in 10% formalin solution (HT501128; Sigma) overnight at 4°C, dehydrated in an ethanol series, and embedded in paraffin wax. 5-μm sections were cut with a microtome and then stained with hematoxylin and eosin for histological analysis.
8
Immunofluorescence Staining of MDCK Cells
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MDCK cells were plated on glass coverslips (#1.5) and allowed to adhere for ∼24 h. Afterward, cells were rinsed twice with PBS (Sigma-Aldrich) and fixed in 4% PFA (HT501128; Sigma-Aldrich) for 15 min at RT. Next, samples were washed three times for 5 min each with PBS and permeabilized with 0.2% Triton-X (in PBS). After three more washes with 1% BSA (A9647; Sigma-Aldrich) in PBS for 5 min each, coverslips were left for >1 h in 1% BSA solution for blocking. Subsequently, primary antibodies against Kapβ1 [ab2811 (3E9); Abcam] and CRM1 (rabbit antibody; kind gift from R. Kehlenbach, University of Göttingen, Göttingen, Germany) in 1% BSA were applied for 1 h at RT. Following another triple-washing step (three times for 5 min each in 1% BSA), the samples were incubated with secondary antibodies: goat anti-mouse Alexa Fluor 568 (A11004; Thermo Fisher Scientific), goat anti-rabbit Alexa Fluor 488 (A11034; Thermo Fisher Scientific), and DAPI (62248; Thermo Fisher Scientific) solution in 1% BSA for 1 h at RT and protected from light. After the last washing step (three times for 5 min each in 1% BSA), coverslips were mounted onto glass slides with VECTASHIELD medium and sealed with nail polish.
9
Immunohistochemical Analysis of Mouse Hearts
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At sacrifice, mouse heart samples were frozen by immersion in isopentane cooled in liquid nitrogen. For immunohistochemistry analysis, heart samples were mounted in optimal cutting temperature compound (OCT) and sectioned on a cryostat at −20 °C to a thickness of 8 μm. Sections were fixed in 10% neutral buffered formalin (SIGMA, HT501128) at RT. After three washes in PBS, samples were permeabilized using 0.2% Triton X- 100 in PBS for 15 min at RT, followed by three washes in PBS, and then blocked in 5% normal goat serum + 2% BSA in PBS for 1 h at RT. After three washes in PBS, samples were incubated with the rabbit anti-HA-Tag (Cell Signaling, 3724) primary antibody 1:2000 in DAKO solution (Agilent, S3022) for 1 h at RT, followed by three additional washes in PBS. Sections were then incubated with goat anti-rabbit IgG (H + L) AlexaFluor diluted 1:300 in DAKO diluent solution 568 for 1 h at RT, washes three times in PBS and finally coverslip with prolonged diamond antifade mountant with DAPI (Thermo Fisher, P36962). All confocal images were acquired using a Zeiss LSM880 microscope using identical acquisition parameters.
10
Histological Analysis of Zebrafish Gut
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Zebrafish gut samples were fixed in 10% neutral-buffered formalin solution (HT501128, Sigma-Aldrich, St. Louis, MO, USA) for 24 h, followed by dehydration, clearing and paraffin embedding. The paraffin-embedded tissues were sectioned at 5 μm in thickness using a microtome. Haematoxylin and eosin (H&E) staining was performed using standard protocols and Alcian blue-periodic acid-Schiff (AB-PAS) staining was conducted following the manufacturer’s instructions (ab245876, Abcam, Cambridge, UK). Primary and secondary antibodies used for immunofluorescent staining were mouse anti-PCNA (sc-56, Santa Cruz Biotechnology, Dallas, TX, USA) and Alexa Fluor® 488 anti-mouse (A11029, Invitrogen, Waltham, MA, USA), respectively. TUNEL assay was conducted using the in situ cell death detection kit (11684795910, Roche, Basel, Switzerland) and augmented by immunostaining using anti-fluorescein CFTM 488A (SAB4600050, Sigma-Aldrich, St. Louis, MO, USA).
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