The left hemisphere of the brain was collected from mice after euthanasia and placed into 10% neutral buffered formalin for 24–48 hours before being transferred to 70% ethanol prior to tissue processing. Samples were processed using a KOS microwave HistoSTATION (Milestone SRL) before being embedded in paraffin wax.
Kos microwave histostation
Manufactured by Milestone
Sourced in Italy
The KOS microwave histostation is a laboratory equipment designed for the rapid processing of tissue samples. It utilizes microwave energy to accelerate the infiltration of paraffin into the tissue, reducing the overall processing time. The device is intended to streamline the histological sample preparation workflow.
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Lab products found in correlation
Fixative solution, by Cell Signaling Technology (1 mentions)
Senescence β galactosidase staining kit, by Cell Signaling Technology (1 mentions)
Paraplast x tra, by Merck Group (1 mentions)
Las af, by Leica (1 mentions)
Fluoromount g, by Thermo Fisher Scientific (1 mentions)
Las af software, by Leica (1 mentions)
Tcs sp8 x, by Leica (1 mentions)
Fish skin gelatine, by Merck Group (1 mentions)
Pt module buffer 1, by Thermo Fisher Scientific (1 mentions)
4 6 diamidin 2 phenylindol dapi, by Roche (1 mentions)
Gill s hematoxylin, by Merck Group (1 mentions)
Ecomount, by Biocare Medical (1 mentions)
Cleaved caspase 3, by Cell Signaling Technology (1 mentions)
8 oxodg, by Merck Group (1 mentions)
Sp7 osterix, by Abcam (1 mentions)
Nanozoomer 2.0 rs, by Hamamatsu Photonics (1 mentions)
Gh2ax, by Merck Group (1 mentions)
Cd146, by Abcam (1 mentions)
8 protocols using kos microwave histostation
1
Mouse Brain Tissue Fixation and Processing
2
Immunofluorescence Staining of Placental Tissues
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Placental tissues and ORGs were fixed in 7.5% formaldehyde and embedded in paraffin. Serial sections (3 µm) of paraffin-embedded material were analyzed by immunofluorescence as described elsewhere (28 (link)). Briefly, sections were deparaffinized in Xylol and rehydrated. Antigen retrieval was performed using 1× PT module buffer 1 (Thermo Scientific) for 35 min at 93 °C using a KOS microwave Histostation (Milestone). Slides were incubated with primary antibodies (listed in SI Appendix, Table S1 ) overnight at 4 °C, washed three times, and subsequently incubated with secondary antibodies (2 µg/mL, 1 h; SI Appendix, Table S1 ). Nuclei were stained with 1 µg/mL DAPI. Tissues were analyzed by fluorescence microscopy (Olympus BX50; CellP Software) and digitally photographed.
3
Paraffin Embedding for Histological Analysis
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After fixation in 4.5% formaldehyde for 48 hours, paraffin embedding was performed using a KOS microwave HistoSTATION (Milestone, Sorisole, Italy) by putting samples in absolute ethanol (35 minutes), followed by isopropanol (70 minutes) and lastly paraffin (90 minutes). Tissue samples were then cut in 5μm sections for histological and fluorescence-based stainings.
4
Senescence β-Galactosidase Assay in Placental Tissues
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SAβG activity was determined in first and third trimester placental and decidual (6th - 12th week of gestation) cryosections using the senescence β-galactosidase staining kit (Cell Signaling) and by adapting published protocols [33 (link), 34 (link)]. Briefly, tissues were preserved in OCT compound, sectioned (4 μm) and fixed in 1x fixative solution (Cell Signaling) for 10 min at room temperature, washed 3 times with PBS and incubated overnight at 37°C with the β-galactosidase staining solution at pH 6.0. Subsequently, slides were counterstained with antibodies against HLA-G, ßG and DAPI. Alternatively, SAßG staining was performed in whole-mount first trimester placentas using the Senescence ß-Galactosidase Staining Kit. Briefly, whole-mount placentas were fixed at 4°C over night with 1x fixative solution, washed 3 times in PBS and incubated over night at 37°C with ß-Galactosidase Staining Solution (pH6). Placentas were subsequently dehydrated and perfused with Paraplast X-TRA (Sigma, St.Louis,MO; USA) using a KOS Microwave Histostation (Milestone, Sorisole; Italy), then embedded in paraffin for serial sectioning and counterstained with antibodies against HLA-G, ßG and DAPI.
5
Placental and Decidual Tissue Immunostaining
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First trimester placental and decidual tissues (6th - 12th week of gestation) were fixed in 7.5% (wt/vol) formaldehyde and embedded in paraffin. Serial sections (3 or 30 μm) were deparaffinised in Xylol (10 min) and rehydrated in a decreasing series of ethanol (100%, 90%, 70%, 0%; 1 min each step). Antigen retrieval was performed using 1× PT module buffer 1 (pH 6, Thermo Fisher Scientific) for 35 min at 93°C using a KOS microwave histostation (Milestone). Sections were blocked using 0.05% cold water fish skin gelatine (Sigma-Aldrich) for 30 min at room temperature and incubated overnight at 4°C with primary antibodies in PBS with 0.05% fish skin gelatine (see antibody list). Secondary antibodies were incubated for 45 min at room temperature in PBS with 0.05% fish skin gelatine and 1 μg/ml 4′,6-Diamidin-2-phenylindol (DAPI, Roche). Finally, tissue sections were mounted with Fluoromount-G (Thermo Fisher Scientific) and covered. Images were acquired with a fluorescence microscope (Olympus BX50 equipped with Cell^P software) or with a confocal laser scanning microscope (LEICA, TCS SP8X, equipped with Leica LAS AF software). Confocal images are depicted as maximum projection of total z-stacks and brightness and contrast were adjusted in a homogenous manner using Leica LAS AF.
6
NHLRC2 Expression in IPF Lungs
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NHLRC2 mRNA in situ hybridization was performed for surgical lung biopsy samples of 8 IPF patients with a histology of UIP (n = 6) or UIP and DAD (n = 2) and in 3 control lung tissue samples using RNAscope 2.5 HD assay—RED and probe Hs-NHLRC2 (555721) according to the manufacturer’s instructions (Advanced cell diagnostics, ACD, Newark, CA, USA). Formalin-fixed and paraffin-embedded specimens were cut into 4 µm thick sections. Target retrieval was performed by boiling the sections at 98 °C for 15 min in RNAscope target retrieval reagent using a KOS Microwave HistoSTATION (Milestone, Sorisole, Italy). Gill’s Hematoxylin (Sigma-Aldrich, St. Louis, MO, USA) was used to stain the nuclei, and coverslips were mounted with EcoMount (Biocare Medical, Pacheco, CA, USA). Positive and negative control probes (Hs-UBC 310041 and DapB 310043, ACD) were used to help qualify samples and control for background noise. Specific staining signals were identified as red dots.
7
Immunohistochemical Analysis of Bone Turnover
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IHC was performed on formalin-fixed, paraffin-embedded 3 μm sections of tumour samples. Immunostaining was conducted using the primary antibodies cleaved caspase-3 (1/400; Cell signalling Technology), CD146 (1/200; Abcam), gH2AX (1/750; Millipore), 8-oxo-dG (1/750; Millipore) and Sp7/Osterix (1/1000; Abcam) on tumour section.
Tibias were decalcified with 4.13% EDTA and 0.2% paraformaldehyde in PBS using the KOS microwave histostation (Milestone) before embedding in paraffin. Sections (Leica Microsystems) were analysed by Osterix and TRAP staining. Quantification of relative OC surface (TRAP+ cells) and OB number (Osterix+ cells) was evaluated by ImageJ (NIH, Bethesda, MD) software. All analyses were assessed by slide scanning using nanozoomer 2.0 RS (Hamamatsu).
Tibias were decalcified with 4.13% EDTA and 0.2% paraformaldehyde in PBS using the KOS microwave histostation (Milestone) before embedding in paraffin. Sections (Leica Microsystems) were analysed by Osterix and TRAP staining. Quantification of relative OC surface (TRAP+ cells) and OB number (Osterix+ cells) was evaluated by ImageJ (NIH, Bethesda, MD) software. All analyses were assessed by slide scanning using nanozoomer 2.0 RS (Hamamatsu).
8
Histomorphometric Analysis of Bone
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Tibias were decalcified with 4.13% EDTA and 0.2% paraformaldehyde in phosphate buffered saline (PBS) for 96 hours using the KOS microwave histostation (Milestone, Kalamazoo, MI) before embedding in paraffin. Sections (3 μm thick, Leica Microsystems) were analyzed by tartrate-resistant acid phosphatase (TRAP) staining as described previously [17, 18] .
Immunostaining for Osterix was performed as described [17] with a rabbit anti-osterix antibody (1/25; Abcam, Cambridge, MA, http://www.abcam.com). Quantification of osteoclast number (TRAP + cells) and osteoblast number (osterix + cells) in the metaphyseal spongiosa was evaluated manually using ImageJ software.in 0.4 mm 2 region of interest (ROI).
For undecalcified histology, tibias were methylmethacrylate-embedded for microtome sectioning (6 μm) for von Kossa staining.
For the dynamic histomorphometry, tibia were dissected and embedded in methylmethacrylate as previously described [3] . Thin sections were mounted unstained and analysed using an image analysis software program (Qwin) interfaced with a microscopy A C C E P T E D M A N U S C R I P T using a DMRXA epifluorescent microscope (Leica). All comparisons of staining intensities were made at 200x magnifications.
Immunostaining for Osterix was performed as described [17] with a rabbit anti-osterix antibody (1/25; Abcam, Cambridge, MA, http://www.abcam.com). Quantification of osteoclast number (TRAP + cells) and osteoblast number (osterix + cells) in the metaphyseal spongiosa was evaluated manually using ImageJ software.in 0.4 mm 2 region of interest (ROI).
For undecalcified histology, tibias were methylmethacrylate-embedded for microtome sectioning (6 μm) for von Kossa staining.
For the dynamic histomorphometry, tibia were dissected and embedded in methylmethacrylate as previously described [3] . Thin sections were mounted unstained and analysed using an image analysis software program (Qwin) interfaced with a microscopy A C C E P T E D M A N U S C R I P T using a DMRXA epifluorescent microscope (Leica). All comparisons of staining intensities were made at 200x magnifications.
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