The alkaline comet assay performed according to the modified OECD TG 489 protocol. Triplicate slides were prepared per treatment. Sperm sample (20 μL) containing 5 × 105 sperm per mL were suspended in 180 μL of 0.5% (w/v) low melting agarose and 55 μL was immediately pipetted onto a comet slide. These slides were incubated for 30 min. The slides were immersed in 250 mL chilled lysis solution with 10 mM DTT for 1 h in a refrigerator. After lysis, the slides were washed twice with DW for 10 min. The slides were then incubated in alkaline buffer (0.3 M NaCl, 1 mM EDTA) for 20 min in the dark at 4°C. Electrophoresis as performed at 21 V and 300 mA for 20 min. After that, the slides were washed twice with DW for 5 min. The slides were then immersed for 5 min in ethanol. Slides were airdried at room temperature in the dark. Immediately before scoring, slides were stained with 55 μL SYBR green (1 : 10,000 dilution of liquid concentrate). Slides were analyzed using a fluorescence microscope (DM-4000B, Leica Microsystems) at 40× magnification. For each sample, 50 comets per slide were analyzed, with three slides scored per sample. The percentage of tail DNA and OTM were measured according to the DNA damage degree using computer software (Komet 5.5, Kinetic Imaging Liverpool) with a fluorescence microscope (DM-4000B, Leica Microsystems).
Dm4000b
Manufactured by Leica
Sourced in Germany, United States, France, Spain, United Kingdom, Canada, Japan, Denmark
The Leica DM4000B is a high-performance microscope designed for advanced laboratory applications. It is equipped with a range of advanced features that enable precise and efficient specimen observation and analysis. The DM4000B's core function is to provide researchers and scientists with a versatile and reliable platform for their microscopy needs.
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Lab products found in correlation
4 6 diamidino 2 phenylindole (dapi), by Merck Group (25 mentions)
Image pro plus 6, by Media Cybernetics (12 mentions)
Mvx10, by Olympus (11 mentions)
Dfc 280, by Leica (9 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (8 mentions)
In situ cell death detection kit, by Roche (8 mentions)
Image pro plus 7, by Olympus (8 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (7 mentions)
Dfc310 fx, by Leica (7 mentions)
4 6 diamidino 2 phenylindole (dapi), by Beyotime (6 mentions)
Las v3, by Leica (6 mentions)
Cw4000 fish, by Leica (6 mentions)
Cm1900, by Leica (6 mentions)
Rm2245, by Leica (5 mentions)
Matrigel, by BD (5 mentions)
Application suite software, by Leica (4 mentions)
Dfc500, by Leica (4 mentions)
Triton x 100, by Merck Group (4 mentions)
Image pro plus 6, by Leica (4 mentions)
Dfc300fx, by Leica (4 mentions)
725 protocols using dm4000b
1
Alkaline Comet Assay for Sperm DNA Damage
2
Histological and Immunohistochemical Evaluation of IVD Degeneration
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After 4% paraformaldehyde fixing for 48 hours, IVD tissue samples were decalcification in the 10% EDTA for 21 days and then embedded into paraffin blocks. After being subjected to 5 μm thickness histological sectioning, hematoxylin and eosin (H&E) staining and safranin O-fast green staining were then processed for histological assessment under standard laboratory protocols. All sections were captured using the high-quality microscope (Leica DM4000B). The histological score was evaluated based on the standard evaluation of 5 categories of degenerative changes [27 (link)]. For immunohistochemical evaluation, tissue sections were first dewaxed by graded xylene and standard alcohol gradients. After washing with PBS and water, 10% goat serum was used to block for 30 min at RT. Subsequently, the sections were incubated with the primary antibodies (anti–MMP9 and anti–MMP13 purchased from Servicebio, Wuhan, China) overnight at 4°C. The next day, the appropriate horseradish peroxidase labeled secondary antibody was incubated with the sections for 1 hour at RT and then developed with diaminobenzidine solution. Finally, all sections were captured under a high-quality microscope (Leica DM4000B).
3
Histological and Immunohistochemical Analysis of DDR1
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Tissues were fixed in 10% formalin, embedded in paraffin, and sectioned at a thickness of 5 μm. Slices were then stained with hematoxylin and eosin (C0105, Beyotime) following the manufacturer’s protocol and visualized using a light microscope (DM4000B, Leica).
Immunohistochemistry for DDR1 was performed as previously described [36 (link), 37 (link)]. Tissue sections were heated with 1X sodium citrate buffer for antigen retrieval in a microwave, incubated with 3% hydrogen peroxide for 10 min to inhibit endogenous peroxidase activity, and blocked in 3% normal goat serum for 1 hour at room temperature. Next, the antibody against DDR1 (ab150506, Abcam, 1:200) was added and incubated overnight at 4°C, followed by the incubation of a secondary antibody (BL003A, Biosharp, 1:1000) at room temperature for 1 h. Sections were then covered with DAB, counterstained with hematoxylin, and mounted. Immunostaining sections were imaged using a light microscope (DM4000B, Leica). Results of immunohistochemistry were quantified by ImageJ software.
Immunohistochemistry for DDR1 was performed as previously described [36 (link), 37 (link)]. Tissue sections were heated with 1X sodium citrate buffer for antigen retrieval in a microwave, incubated with 3% hydrogen peroxide for 10 min to inhibit endogenous peroxidase activity, and blocked in 3% normal goat serum for 1 hour at room temperature. Next, the antibody against DDR1 (ab150506, Abcam, 1:200) was added and incubated overnight at 4°C, followed by the incubation of a secondary antibody (BL003A, Biosharp, 1:1000) at room temperature for 1 h. Sections were then covered with DAB, counterstained with hematoxylin, and mounted. Immunostaining sections were imaged using a light microscope (DM4000B, Leica). Results of immunohistochemistry were quantified by ImageJ software.
4
Histometric Analysis of Alveolar Bone Loss
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After radiographic and tomographic analysis, the maxillae were decalcified in buffered (pH 8.0) 17% EDTA (Sigma Chemical Co, St Louis, MO). After the decalcification period, samples were histologically processed for paraffin embedding. Serial sections (6 µm) were cut and stained with hematoxylin and eosin for analysis under an optical microscope (DM 4000B, Leica, Wetzlar, Germany).
For each sample, the central histologic section containing the CEJ, periodontal tissues, and the alveolar bone were histometrically measured by a single calibrated histologist, blinded to the groups, using an image processing system, which consisted of a light microscope (DM 4000B, Leica, Wetzlar, Germany), a color camera (DFC 500, Leica), a color image processor (Leica Qwin V3 software, Leica) and a personal computer (Intel Corel i7, 3.4 GHz, Windows 7 Ultimate). The alveolar bone loss was assessed by measuring the linear distance between the CEJ and the buccal alveolar bone crest, in the central histologic sections from the side of the maxilla (Fig. 1 c).
For each sample, the central histologic section containing the CEJ, periodontal tissues, and the alveolar bone were histometrically measured by a single calibrated histologist, blinded to the groups, using an image processing system, which consisted of a light microscope (DM 4000B, Leica, Wetzlar, Germany), a color camera (DFC 500, Leica), a color image processor (Leica Qwin V3 software, Leica) and a personal computer (Intel Corel i7, 3.4 GHz, Windows 7 Ultimate). The alveolar bone loss was assessed by measuring the linear distance between the CEJ and the buccal alveolar bone crest, in the central histologic sections from the side of the maxilla (
5
Chorioallantoic Membrane Assay for Toxicity
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The CAM technique was performed to measure the toxicity, biocompatibility and antiangiogenic activity of LyeTx I b on 72 eggs (n = 12 for each group) [17 (link)]. The procedure has been found to be an acceptable alternative to in vivo tests and was performed according to [17 (link)] with minor modifications. Fertilized eggs were purchased from Rivelli (Igarapé Brazil) and placed in a rotating incubator in a humidified atmosphere at 37 °C until testing on day 5. The shell above the air cell of the eggs and the inner membrane were removed using forceps and the CAM was assessed. LyeTx I b (0.7 and 2.89 μM) was applied directly onto the CAM which was then examined for 72 h by obtaining a photo with a light microscope (Leica, model DM4000B, Germany) coupled to a Leica digital CCD camera model DFC 280 (Software Leica Application Suite V 3.3.0, Germany) illumination (Leica, model DM4000B, Germany). Each concentration of LyeTx I b was tested 12 times and the experiment was repeated once. Neovascularization was measured using the software Image J. Densitometric and nonsaturated vessels were analyzed according to the number of pixels.
6
Histological Characterization of Implants
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For histology, after 2 weeks implantation, some implants were fixed for 24 h in Bouin-Hollande and embedded in paraffin. Serial sections (7 µm) were stained with Mallory's stain. Sections were observed in a Leica DM4000B microscope. Other implants were embedded in Tissue-Tek, frozen at -20°C and sectioned (10µm) using a cryostat (Leica, CM3000). Serial sections were rinsed with PBS and fixed for 10 min with 4% paraformaldehyde at 4°C. After washing three times for 5 min in PBS at room temperature, sections were incubated for 30 min at room temperature in a blocking solution of 1% bovine serum albumin (BSA) and 0.1% Triton X100 and then incubated for one night at 4°C with the primary antibodies (Table 1). After washing with PBS, sections were incubated with secondary antibodies (Molecular Probes, Invitrogen) (Table 1). Nuclei were stained with 4', 6-diamidino-2-phenylindole (DAPI, Euromedex, Souffelweyersheim, France). Negative controls were performed either omitting the primary antibody or using normal goat serum (NGS). After 3 additional washes in PBS, slides were mounted in fluorescence mounting medium (Dako, Trappes, France) and observed with a fluorescence microscope (Leica DM4000B).
7
Comprehensive GPC2 Expression Analysis in TMAs
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Formalin-fixed, paraffin-embedded TMA sections were analyzed for GPC2 expression at both CHOP and BC using GPC2 antibody (Santa Cruz). At CHOP, staining was performed on a Bond Max automated staining system (Leica Biosystems) using the Bond Refine polymer staining kit (Leica Biosystems).27 (link) At BC, tissue sections were incubated in Tris EDTA buffer (cell conditioning 1; CC1 standard) at 95°C for 1 hour to retrieve antigenicity, followed by incubation with GPC2 antibody at 1:100 for 1 hour. Intensity scoring was performed on a common four-point scale: 0: no staining, 1: low but detectable degree of staining, 2: clearly positive staining, and 3: strong staining. Expression was quantified as H-Score, the product of staining intensity and % of stained cells. TMA scoring included both tumor and stromal cells. TMAs were scored by two methods to ensure accurate results including a blinded pathologist (one at CHOP, one in British Columbia) and Aperio RUO Image Analysis by Aperio ImageScope. Photographs were taken using a Leica DM4000B.
Xenograft animals treated with CAR T cells had tumors excised and were also stained with GPC2 (Santa Cruz), H&E, human nuclei stain (Abcam), and human CD3 (Dako) per protocol:https://www.research.chop.edu/sites/default/files/web/sites/default/files/pdfs/Antibody_Protocols.pdf . Photographs were taken using a Leica DM4000B.
Xenograft animals treated with CAR T cells had tumors excised and were also stained with GPC2 (Santa Cruz), H&E, human nuclei stain (Abcam), and human CD3 (Dako) per protocol:
8
Microscopic Examination of Rust Fungus Development
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Nuclei were stained with DAPI (Sigma Chemical Company, St. Louis, MO, USA) and were dated under fluorescence microscope (Leica DM4000B, Leica Microsystems GmbH, Wetzlar, Germany). Urediniospores of ΔTs06 were prepared in spore suspensions at a concentration of 0.01 g/ml and were smeared on a 2% water agar medium for germination and on abaxial leaves of P. purdomii for inoculation as described in Zheng et al. (2019 (link)). Histological observation of intercellular hyphae, haustorial mother cells, and haustoria was implemented using a Hitachi HT-7700 TEM (Hitachi, Tokyo, Japan) (Kang and Buchenauer, 2000 (link)). Urediniospores of ΔTs06 were continuously subcultured on P. purdomii and harvested five times, and the proportion of polykaryotic urediniospores was counted in each harvest. Meanwhile, P. purdomii of 10 days post inoculation (dpi) were transferred to a chamber to induce teliospores at 4°C, and basidiospores developed according to the protocol outlined in Yu et al. (2009 ). Thereafter, basidiospores were inoculated on the needles of a larch, and then they were moved to the greenhouse until spermatia developed (Pernaci et al., 2014 (link)). The basidia, basidiospores, spermatia, and aeciospores were stained using DAPI and were observed and photographed using a Leica DM4000B microscope. The strain Ts06, used as a control, was observed simultaneously with ΔTs06.
9
Histological Analysis of Myocardial Fibrosis
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The rats were anesthetized by intramuscular injection of a mixture of zoletil 50 (30 mg/kg) and rompun (10 mg/kg). Rats were weighed and their hearts were removed and divided into two halves along the anterior longitudinal middle line. One half of each heart was fixed in formalin, embedded in paraffin, and cut into 4 µm thick sections. The other half was frozen in liquid nitrogen and stored at −80℃ for real-time polymerase chain reaction (PCR) and Western blot analyses. The extent of myocardial fibrosis was determined by visualizing fibrotic tissue using Masson's trichrome (MT) staining. Apoptotic cardiomyocytes were evaluated using the terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay in paraffin sections with an In Situ Cell Death Detection kit (Roche, Mannheim, Germany). The stained sections were photographed using a light microscope (Leica DM 4000B; Leica, Wetzlar, Germany). Five regions from each digitized images were selected at random from the individual sections and quantified using the Leica image analysis system (Leica DM 4000B). All data were evaluated by an independent blinded investigator.
10
Evaluating Gene Therapy Effects on Corneal Anatomy
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To study the effect of gene delivery on corneal anatomy, rabbit corneal tissue sections from the naïve and AAV5-naked, and AAV5–Dcn groups were subjected to hematoxylin and eosin (H&E) and Mason trichome staining following our published protocols.25 (link),26 (link) The H&E and Mason trichome stained corneal sections were imaged with a bright-field microscope (Leica DM 4000B, Leica Microsystems Inc.) equipped with a digital camera and imaging software (SpotCamRT KE; Diagnostic Instruments, Sterling Heights, MI). The effect of gene therapy on cellular density was studied by mounting corneal sections with an antifade Vectashield mounting medium with propidium iodide (PI) (Vector Laboratories, Inc., CA). The PI-stained nuclei in corneal sections were recorded with a fluorescence microscope (Leica DM 4000B, Leica Microsystems Inc.) equipped with a digital camera (SpotCam RT KE, Diagnostic Instruments Inc.). The cellular density in the corneas of the naïve, AAV5 naked, and AAV5–Dcn groups was quantified by counting PI-stained nuclei27 (link) in ten randomly selected areas in corneal sections at 200 and/or 400 magnification field following the method reported by our laboratory earlier.21 (link)
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