Then, a midline laparotomy was performed and all sections of the gastrointestinal tract were removed. Approximately 1 cm fragments of the jejunum (approximately 20 cm from the duodenum) from each animal was collected for further research. Tissue were subjected to the standard immunofluorescence frozen procedure by Makowska et al. as previously described [25 (link)]. Collected jejunum fragments were placed in a 4% buffered solution of paraformaldehyde (pH 7.4). Immersion fixation time was 1 h. Subsequently, samples were transferred to a phosphate buffer solution (PBS, pH 7.4) for 72 h (a buffer was exchanged 3 times, every 24 h). After this time, the collected jejunum were dehydrated into an 18% buffered sucrose solution for two weeks. After this time, frozen blokes were performed. The tissue blocks were cut in frontal or sagittal planes by means of a Microm HM 560 cryostat (Carl Zeiss, Berlin, Germany) at a thickness of 14 μm and attached on gelatinized glass slides suitable for immunohistochemistry.
Microm hm 560
Manufactured by Zeiss
Sourced in Germany
The Microm HM 560 is a cryostat designed for sectioning frozen tissue samples. It features a temperature range from -10°C to -50°C and a section thickness range of 1 to 100 micrometers. The instrument is equipped with a motorized specimen feed and a manual trim function.
Automatically generated - may contain errors
Lab products found in correlation
Axioplan microscope, by Zeiss (2 mentions)
Vetbutal, by Biowet (1 mentions)
Hexane, by Fujifilm (1 mentions)
Basms 2325, by Fujifilm (1 mentions)
Bas 5000, by Fujifilm (1 mentions)
Optimal cutting temperature compound, by Sakura Finetek (1 mentions)
Adhesive silane coated glass slide, by Matsunami (1 mentions)
6 protocols using microm hm 560
1
Immunofluorescence Analysis of Jejunum Tissue
2
Radionuclide-Induced Tumor Damage Evaluation
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To investigate 211At-AITM-induced damage events, MDA-MB231 (2 × 105 cells) and MIA PaCa2 cells (2 × 105 cells) were exposed to 18.5 kBq/mL 211At-AITM or vehicle (PBS) for 21 h in triplicate in 24-well plates. Alterations in cell morphology, viability, and cell cycle distribution were subsequently analyzed until 7 days or otherwise indicated. Animals were anesthetized with isoflurane and then sacrificed. Tissue samples of xenograft tumors were harvested and frozen at 2, 7, and 30 days after 211At-AITM injection. Tumor sections (5 μm) were prepared using a Microm HM560 microtome (Carl Zeiss Jena, Germany).
3
Immunostaining of Muscle Proteins
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Immunostaining of ADBR2, MuSK and α -dystroglycan was performed on 10 μm transverse sections of gastrocnemius muscle cut using a cryostat (Microm HM 560, Zeiss) in the region of the motor end-plate. Sections were fixed in acetone at 4 °C for 15 minutes and then permeabilised in 0.1% Triton X-100 for 15 minutes at room temperature. Sections were blocked in PBS containing 10% goat serum, 1% BSA for 30 minutes and then incubated with primary antibody (rabbit anti-MuSK 1:500, Abcam; rabbit anti-ADRB2 1:200; Santa Cruz Biotechnology; mouse anti-α -dystroglycan 1:100; Santa Cruz Biotechnology) in blocking buffer for 2 hours at room temperature. Sections were washed and incubated in Alexa Fluor® 488 α -BTX (1:250), and secondary antibodies Alexa Fluor® 594 goat anti-rabbit (1:250) for 1 hour at room temperature. Sections were then washed and mounted using Vectashield mounting medium.
4
Transcardial Perfusion and Cryosectioning
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Six weeks after streptozotocin injection, animals were deeply anesthetized via intravenous administration of pentobarbital (Vetbutal, Biowet, Poland) and perfused transcardially via the ascending aorta with 4% paraformaldehyde in a 0.1 mol/L phosphate buffer (PB, pH 7.4). The samples were post-fixed by immersion in the same fixative for 1 h, rinsed several times with phosphatase buffer (PB) and then transferred into 30% sucrose solution and stored at 4 °C until sectioning. The tissue blocks were cut in frontal or sagittal planes using a Microm HM 560 cryostat (Carl Zeiss, Germany) at a thickness of 12 μm and mounted on gelatinized glass slides.
5
Muscle Histological Analysis
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Transverse 10 µm sections of TA, intercostal, soleus and EDL muscles were cut using a cryostat (Microm HM 560, Zeiss). Sections were stained with haematoxylin and eosin according to standard procedures. Slides were mounted with DPX Mounting Medium (LAMB) and images were acquired using a Zeiss Axioplan microscope and AxioVision software.
6
Cardiac Tissue Extraction and Imaging
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MI model rats were sacrificed under ether anesthesia. Representative cardiac tissue specimens were harvested immediately, embedded in optimal cutting temperature compound (Sakura Finetek USA, Torrance, CA, USA), and frozen in hexane (Wako Pure Chemical Industries, Osaka, Japan). The sections (5 μm) were prepared using a cryotome, MICROM HM560 (Carl Zeiss, Jena, Germany), at a temperature of − 20 °C and mounted on adhesive silane-coated glass slides (Matsunami Glass Industries, Kyoto, Japan). In accordance with the established procedure [21 (link)], the sections were then pre-incubated in 50 mM Tris buffer (pH 7.4) at room temperature for 20 min followed by incubation in the same buffer containing [18F]FEDAC (18 MBq/L) at room temperature for 30 min. After incubation, the sections were washed twice for 2 min each in 50 mM cold fresh Tris–HCl buffer and for 10 s in distilled water. They were then dried with a warm air current, placed in contact with an imaging plate (BAS-MS 2325; Fujifilm, Tokyo, Japan) for 60 min, and analyzed using a Bio Imaging Analyzer System (BAS 5000; Fujifilm).
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