MSC-embedded scaffolds (in vitro) and extracted MSC-embedded scaffolds (in vivo) were fixed in 0.1 M NBF for 24 h, followed by decalcification in ethylenediaminetetraacetic acid (EDTA) solution for one week, until no traces of calcified tissue remained. Specimens were then dehydrated in graded ethanol solutions (70%, 95%, and 100%), embedded in paraffin, and then cut with a microtome to 7 μm thick histological sections. Finally, the samples were stained with hematoxylin and eosin (H&E), to identify the nucleus and the connective tissue. Histological slides were scanned using an automatic digital slide scanner (3D Histech Pannoramic MIDI, 3DHISTECH) and the percentage of the newly formed bone was quantified using the panoramic viewer software (3DHISTECH.).
3d histech pannoramic midi
Manufactured by 3DHISTECH
Sourced in Hungary
The 3D Histech Pannoramic MIDI is a high-performance digital slide scanner designed for use in digital pathology applications. The device captures digital images of tissue samples mounted on microscope slides, enabling efficient and accurate analysis of specimens.
Automatically generated - may contain errors
Lab products found in correlation
Panoramic viewer software, by 3DHISTECH (1 mentions)
Ab2219, by Merck Group (1 mentions)
Ab97051, by Abcam (1 mentions)
Alexa fluor conjugated secondary antibody, by Thermo Fisher Scientific (1 mentions)
Tissuefax, by TissueGnostics (1 mentions)
Sudan black, by Merck Group (1 mentions)
Fluoview fv10i confocal microscope, by Olympus (1 mentions)
Pannoramic viewer, by 3DHISTECH (1 mentions)
Romulin aec chromogen kit, by Biocare Medical (1 mentions)
Ab14106, by Abcam (1 mentions)
Ab211326, by Abcam (1 mentions)
Ab203196, by Abcam (1 mentions)
Ab95824, by Abcam (1 mentions)
5 protocols using 3d histech pannoramic midi
1
Quantitative Histological Analysis of MSC-Embedded Scaffolds
2
Epididymal AQP1 and AQP9 Expression
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Epididymal sections from the LP and NP animals (sections of animals from different litters; n = 4 animals/group) were subjected to antigen retrieval in a humid environment (electric pot) at 100 °C in Tris/0.1 M EDTA pH 9.0 for 30 min. After being washed in distilled water, the sections were subjected to the blocking of endogenous peroxidase (3% hydrogen peroxide in methanol) for 15 min. To block nonspecific binding, the slides were incubated with 3% skim milk in PBS for 1 h. Then, the sections were incubated overnight (at 4 °C) with primary antibodies to AQP1 (concentration 1:200; AB2219 EMD-Millipore Corp.®, Billerica, MA, USA) or AQP9 (concentration 1:200, APQ91-A Alpha Diagnostic, San Antonio, TX, USA), which were diluted in 1% BSA. After incubation with the primary antibodies, the sections were washed in PBS and then incubated with anti-rabbit secondary HRP antibody (ab97051; concentration 1:200, Abcam Inc.®, Cambridge, MA, USA) for 2 h at room temperature. The reaction was visualized with DAB chromogen (3,3′-diaminobenzidine tetrahydrochloride; Sigma-Aldrich Co.®, St. Louis, MO, USA) and counterstained with hematoxylin for 1 min. The slides were scanned using a 3D Histech Pannoramic MIDI (3DHistech, Budapest, Pest, Hungary) and analyzed and photographed using the Pannoramic Viewer program.
3
Immunostaining of Mouse Kidney Tissue
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Mouse kidneys were fixed with 4% paraformaldehyde, embedded in paraffin, sectioned, and stained with standard immunohistochemistry and fluorescent microscopy methods as previously described20 (link),7 (link). An additional antigen retrieval step was applied in all experiments by heating samples in a Tris-based buffer (pH 9.0) to 95 °C for 20 min. Primary antibodies are listed in Supplementary Table S2 . Alexa Fluor-conjugated secondary antibodies were purchased from ThermoFisher. Slides were incubated with 1% Sudan black (Sigma-Aldrich) in 75% ethanol at room temperature for 20 min to reduce tissue auto-fluorescence before mounting. Confocal images were captured using an Olympus FLUOVIEW FV10i confocal microscope. Scanning of the immunofluorescent images of the whole kidney tissue was performed using TissueFAXS (TissueGnostics). For quantification of urothelium thickness, paraffin sections were stained with haematoxylin and eosin. Bright-field images were scanned using 3D Histech Pannoramic MIDI (3DHISTECH). The average urothelium thickness of the UPJ was calculated using Pannoramic viewer (3DHISTECH).
4
Immunohistochemistry Protocol for Paraffin Sections
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Paraffin-embedded sections were deparaffinized and rehydrated. Antigen retrieval was performed by boiling the sections in sodium citrate solution in a pressure-cooker for 3 min. Endogenous peroxidase was blocked by 3% H2O2 solution for 10 min at 37°C and washed with PBST. Then, the paraffin sections were incubated with the primary antibody at 4°C overnight. The next day, the paraffin sections were rewarmed at 37°C for 10 min. After two washes in PBS and treatment with an adjuvant for 20 min at 37°C, the sections were incubated with the second antibody for 30 min at 37°C. The immunohistochemical reaction was visualized with 3,3,0-diaminobenzidine (DAB) for 3 min. Sections were counterstained for 3 min in hematoxylin (zSGB-Bio, Beijing). Slides were scanned with the digital 3DHISTECH–Pannoramic MIDI (3DHISTECH Ltd, Budapest). The image analysis software ImageJ IHC Profiler was used for staining quantification (24 (link)).
5
Immunohistochemical Analysis of Lung Tissue
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Lung tissues were embedded in paraffin, cut into 4 μm sections, and stained with hematoxylin and eosin (H&E). Inflammatory cell infiltration and lung architecture were assessed by light microscopy. The mucus secretion level was detected by periodic acid-Schiff (PAS; Sigma-Aldrich) staining. Lung sections were deparaffinized, hydrated in water, and then stained with periodic acid for 5 min. For immunohistochemistry, samples were paraffin embedded, sectioned, and stained by standard immunohistochemistry methods as previously described (24 (link), 25 (link)). An additional antigen retrieval step was applied by heating samples in a Tris-based buffer (pH 9.0) to 95°C for 20 min. After blocking in 5% bovine serum albumin for 30 min at room temperature, tissues were incubated overnight at 4°C with primary antibodies against IL-19 (1:100; ab14106, Abcam), IL-20R1 (1:100; ab203196, Abcam), IL-20R2 (1:100; ab95824, Abcam), and SPC (1:100; ab211326, Abcam). The immune-reactivity of positive staining was developed by using the AEC chromogen kit (Romulin AEC Chromogen Kit; Biocare Medical, Walnut Creek, CA) and counterstained with Mayer's hematoxylin (J. T. Baker, Phillipsburg, NJ). Images were captured using an immunofluorescence microscope (Olympus BX51, Tokyo, Japan) or scanned using 3D Histech Pannoramic MIDI (3DHISTECH).
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