The left femurs and the fifth lumbar vertebra of mice were decalcified in 10% EDTA solution for 28 days and the solution was renewed every 2 days. Femurs and other organs were dehydrated through automatic dehydrator (Microm STP120, Thermo). After embedded in paraffin, the tissues were sectioned at 4 μm thickness by rotary microtome (Microm HM325, Thermo). Then the sections were dewaxed in dimethylbenzene and graded ethanol, then immersed in water for 2 min. The sections were stained with Haematoxylin solution (BBI, #E607318-0206) for 1 min, differentiated in PBS for 5 min, then stained with Eosin solution (BBI, #E607318-0206) for 10 sec and rinsed with water. The sections were mounted with neutral resin after 10 min vitrification by dimethybenzene. Pictures were taken using the inverted microscope (DS-Ri1, Nikon).
Microm stp 120
Manufactured by Thermo Fisher Scientific
Sourced in Germany, United States
The Microm STP 120 is a tissue processor used for the dehydration, clearing, and paraffin infiltration of tissue samples. It is designed to automate the sample preparation process prior to sectioning and staining for microscopic analysis.
Automatically generated - may contain errors
Lab products found in correlation
Masson trichrome, by Merck Group (2 mentions)
Microm hm 325, by Thermo Fisher Scientific (2 mentions)
Fluoromount g mounting media, by Southern Biotech (2 mentions)
Dapi nuclear stain, by Southern Biotech (2 mentions)
Ds ri1, by Nikon (1 mentions)
Alexa fluor 488 conjugated wga, by Thermo Fisher Scientific (1 mentions)
Clearvue coverslipper, by Thermo Fisher Scientific (1 mentions)
Mason s trichrome, by Merck Group (1 mentions)
Naoh pellet, by Merck Group (1 mentions)
Rm2255, by Leica (1 mentions)
Immpress hrp reagent kit, by Vector Laboratories (1 mentions)
Microtome, by Leica (1 mentions)
Histostar workstation, by Thermo Fisher Scientific (1 mentions)
Vitrogel, by BioVitrum (1 mentions)
Finesse me microtome, by Thermo Fisher Scientific (1 mentions)
Axiocam mrc5 camera, by Zeiss (1 mentions)
Perfection v600 scanner, by Epson (1 mentions)
Histomix, by BioVitrum (1 mentions)
Histostar, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 488 conjugated wheat germ agglutinin, by Thermo Fisher Scientific (1 mentions)
11 protocols using microm stp 120
1
Histological Analysis of Mouse Femurs and Vertebra
2
Cardiac Fibrosis and Structure Analysis
3
Histological Assessment of Liver Tissues
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Preserved samples of liver tissues were processed for histological analysis, using an automated tissue processor (Microm STP 120 Thermo Scientific, Waltham, MA, USA), and embedded in paraffin wax. The liver samples were then sectioned at 5 µm, using a microtome (Leica instruments GmbH, (Pty) Ltd., Wetzlar, Germany), and then mounted on glass slides which were stained automatically with hematoxylin and eosin (H&E) or Mason’s trichrome (MT) in a Gemini AS slide stainer coupled to a Clearvue coverslipper (Thermo Scientific, Massachusetts, USA). The liver H&E-stained slides were used to ascertain hepatocellular changes, while the MT stain was used to assess collagen deposition as a marker of fibrosis.
The H&E- and MT-stained liver slides were viewed under a light microscope (Olympus XC 10 HD, Glasgow, UK) mounted on an Olympus BH2-RFCA camera and (Carl Zeiss Microscopy GmbH, Göttingen, Germany), respectively, to evaluate the histological changes in the liver.
The H&E- and MT-stained liver slides were viewed under a light microscope (Olympus XC 10 HD, Glasgow, UK) mounted on an Olympus BH2-RFCA camera and (Carl Zeiss Microscopy GmbH, Göttingen, Germany), respectively, to evaluate the histological changes in the liver.
4
Histological Analysis of Plaque Samples
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The plaque samples were fixated in 3.7% formalin solution for 24 h. Next, the samples were decalcified in buffered ethylenediaminetetraacetic acid (EDTA) solution for a week. The buffered EDTA solution was prepared by mixing 400 ml phosphate buffered saline (PBS) solution with 40 g of ETDA powder (Sigma–Aldrich Chemie GmbH, Munich, Germany). Next, NaOH pellets (Merck, Darmstadt, Germany) were added gradually to adjust the pH to 7.5.
Next, tissue samples were dehydrated with a standard tissue processor (Microm STP 120, Thermo Fisher Scientific, Walldorf, Germany) and embedded in paraffin blocks. Samples were sectioned in the transverse plane with the thickness of 5 μm using a microtome (RM2255, Leica Microsystems B.V., Amsterdam, The Netherlands). Finally, histology sections were stained with Weigner's Iron Hematoxylin and Mason's trichrome (Sigma–Aldrich Chemie GmbH, Munich, Germany). The histology overview images were manually matched to the US images based on the structural similarity of the lumen and vessel wall.
Next, tissue samples were dehydrated with a standard tissue processor (Microm STP 120, Thermo Fisher Scientific, Walldorf, Germany) and embedded in paraffin blocks. Samples were sectioned in the transverse plane with the thickness of 5 μm using a microtome (RM2255, Leica Microsystems B.V., Amsterdam, The Netherlands). Finally, histology sections were stained with Weigner's Iron Hematoxylin and Mason's trichrome (Sigma–Aldrich Chemie GmbH, Munich, Germany). The histology overview images were manually matched to the US images based on the structural similarity of the lumen and vessel wall.
5
Immunohistochemical Analysis of Vascular Tissues
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The blood vessel rings were placed into 4% paraformaldehyde, left overnight, and then transferred into 1× phosphate buffered saline (PBS) at 4 °C. This was followed by paraffin processing (Microm STP120, Thermo Scientific, Waldorff, Germany) and embedding in paraffin blocks. Sections were cut at 5 μm, deparaffinised in xylene, rehydrated, and blocked with 1% goat serum in 10 mm TrisCl (pH 7.4) for 20 min. Primary mouse monoclonal anti-bodies Anti-3-Nitrotyrosine [39B6] (Abcam 61392) and eNOS type III (BD Biosciences 610296) at 1:100 dilution were applied overnight. A no primary antibody control was completed to detect non-specific protein binding. Samples were subsequently incubated with anti-mouse IgG for 1 h (Immpress HRP reagent kit, MP-7452 Vector laboratories). Diaminobenzidine (DAB) (BD Biosciences 550880) was applied as a chromogen before counterstaining with hematoxylin, dehydration, and mounting in Dibutylphthalate Polystyrene Xylene (DPX) [24 (link)].
6
Gastric Biopsy Processing for Histology
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Gastric biopsies were processed with a spin tissue processor (MicromSTP120, ThermoScientific Corp., Walldorf, Germany) comprising the following steps: Formol immersion (2 h), dehydration in alcohol 96% (6 h), alcohol 100% (4 h) and xylene (3 h), and paraffin immersion at 56-58 °C (3 h) and at 62 °C (3 h). Samples were then embedded in paraffin at 62 °C, from which 4 μm consecutive sections were obtained for haematoxylin-eosin and Giemsa histologic staining. Microscopic assessment was classified according to the updated Sydney System Classification[31 (link)].
7
Histological Analysis of Liver Tissue
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Liver tissue samples preserved in 10% phosphate-buffered formalin were processed with an automated tissue processor (Microm STP 120 Thermo Scientific, Waltham, MA, USA), embedded in paraffin wax and sectioned at 3 µm using a microtome (Leica instruments GmbH, (Pty) Ltd., Wetzlar, Germany) for histological analysis. The sections were stained with haematoxylin and eosin (H&E) using a Gemini AS slide stainer coupled to a Clearvue cover slipper (Fisher Scientific, Waltham, MA, USA). The stained liver sections were viewed under an Olympus BH2-RFCA microscope (Olympus Corporation, Tokyo, Japan) coupled to an Olympus XC 10 HD camera (Olympus Corporation, Tokyo, Japan) for histological assessment and image capture.
8
Histological Examination of Formalin-Fixed Tissues
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Formaldehyde-fixed samples were dehydrated in a graduated series of alcohols at increasing degrees, from 70° to 100°, using a circulating inclusion automaton (Thermo Scientific, Microm STP 120, UK) and incorporated in kerosene (paraffin). Paraffin sections (5 µm thick) were mounted on a clean glass slide and stained with hematoxylin and eosin (H&E) (BioLab, Mumbai, India) for light microscopic examination (×40) (Leica DMLB, Wetzlar, Germany) [25 ].
9
Assessing Lung Pathology in Mice
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Lungs from each mouse were fixed in 10% neutral buffered formalin (Soluformtm, JLS-Chemical, Russia) at +4°C, dehydrated in isoprepe (BioVitrum, Russia) using Microm STP 120 (Thermo Scientific, USA), and embedded in HISTOMIX (BioVitrum, Russia) using HistoStar workstation (Thermo Scientific, USA). 5-μm thick sections (10 per mouse) were cut using a Finesse ME+ microtome (Thermo Scientific, USA), stained with haematoxylin and eosin and mounted in Vitrogel (all BioVitrum, Russia). Pictures were obtained either using an Epson Perfection V600 scanner (9600 dpi) or using an Imager. Z1 microscope with an AxioCam MRc 5 camera (all ZEISS, Germany) at 20х and 63х magnification. Pathological changes were assessed using three parameters: (1) the acute lung injury (ALI) score, showing mostly the immunopathology in the lung parenchyma (from 0 to 1); (2) the peribronchiolar infiltration score, estimating the degree of inflammation of the tissue around the bronchioles (from 0 to 5); and (3) the perivascular infiltration score, estimating the degree of inflammation of the tissue around vessels (from 0 to 5). The exact parameters of tissue scoring systems are provided in the Supplementary Materials (p. 9).
10
Histological Analysis of Cardiac Fibrosis
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Subgroups of hearts were harvested at 4 and 14 weeks post-TAC for analyses of structure, fibrosis, and biochemistry. Trichrome staining was performed as previously described [9] (link). Briefly, mice were euthanized and following cardioperfusion, hearts were fixed for 1-3 days in 4% paraformaldehyde at 4°C. Hearts were dehydrated and paraffinized using a Microm STP 120 from ThermoFisher Scientific, embedded in paraffin using a HistoStar apparatus (ThermoFisher), and sectioned (4-6 micron) using a Microm HM 325 (ThermoFisher). Tissue sections were then stained with Weigert's iron hematoxylin and Masson Trichrome (Sigma-Aldrich) according to the manufacturer's instructions. Interstitial fibrosis was quantified by color threshold measures using ImageJ. Alternatively, tissue sections were de-paraffinized and re-hydrated according to the Trichrome staining protocol, but following the wash with de-ionized water heart sections were washed 3 times 5 minutes with 1x PBS followed by staining with Alexa Fluor 488-conjugated wheat germ agglutinin (WGA, 1:10 in 1xPBS) (Invitrogen) for 1 hour at room temperature in a humidified chamber in the dark. Sections were again washed 3 times 5 minutes with 1x PBS followed by mounting with coverslips using Fluoromount-G mounting media containing DAPI nuclear stain (Southern Biotech).
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