Uterine sections were prepared as previously described [156 (link)]. Briefly, slides were dried at 55 °C for 30 min, deparaffinized, cleaned using the Sub-X Cleaning Medium (Leria SUB-X 3803670, Carterville, IL, USA), and hydrated in various concentrations of ethanol. Antigen retrieval was performed using citrate buffer (K-035; Diagnostic Biosystems, København, Denmark) at pH 6.0 via autoclave heat treatment. After inhibiting endogenous peroxidase (ab64218; Abcam, Cambridge, UK) for 15 min at room temperature, slides were incubated with TGF-β (bs-4538R; BIOSS, Massachusetts, USA) at 4 °C overnight. The secondary antibody was applied using DAKO REALTM EnVision (DAKO K5007, Silkeborg, Denmark) and incubated for 30 min at room temperature, followed by Rabbit/Mouse (DAB+) incubation for 5 min. The slides were counterstained with hematoxylin (105175; Merck, Millipore, MA, USA) for 10 min and mounted using CC/Mount (C9368; Sigma-Aldrich, Louis MO, USA). Throughout the experiments, thorough washing was performed with TBS-T. Scanning was conducted using the panoramic scanner (Sysmex Europe GmbH, Norderstedt, Germany), and images were captured using a microscope (Sysmex TOA Medical Electronics, Europe) GmbH, Hamburg, Germany). Finally, results were quantified using the ImageJ software version 1.8.0. (National Institutes of Health, Bethesda, ML, USA).
Cc mount
Manufactured by Merck Group
Sourced in United States
The CC/Mount is a type of lens mount designed for use with compact and camera-mounted microscopy equipment. It provides a standardized interface for attaching compatible lenses, cameras, and other accessories to enable microscopic imaging and analysis.
Automatically generated - may contain errors
Lab products found in correlation
Zen blue software, by Zeiss (2 mentions)
Lsm 700, by Zeiss (2 mentions)
Ab64218, by Abcam (1 mentions)
Hematoxylin, by Merck Group (1 mentions)
Neutral buffered formalin solution, by Merck Group (1 mentions)
Luciag image analysis system, by Nikon (1 mentions)
Eclipse e800, by Nikon (1 mentions)
Donkey serum, by Merck Group (1 mentions)
Superpicture 3rd gen ihc detection kit, by Thermo Fisher Scientific (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions)
Vectastain abc ap staining kit, by Vector Laboratories (1 mentions)
Meyer s hematoxylin, by Merck Group (1 mentions)
Vectamount, by Vector Laboratories (1 mentions)
Casein, by Merck Group (1 mentions)
Immpact dab substrate kit, by Vector Laboratories (1 mentions)
Axioplan 2, by Zeiss (1 mentions)
Ab2426, by Abcam (1 mentions)
A11008, by Thermo Fisher Scientific (1 mentions)
Ab15580, by Abcam (1 mentions)
Alexa fluor 488 conjugated goat anti rabbit igg h l secondary antibody, by Thermo Fisher Scientific (1 mentions)
20 protocols using cc mount
1
Immunohistochemical Analysis of TGF-β in Uterine Tissue
2
Oil Red O Staining of RCC Tissue
3
Immunohistochemical and Immunofluorescent Staining
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Paraffin-embedded 5 mm tissue sections were deparaffinized, and antigen retrieval was performed using sodium citrate retrieval solution (Sigma). Immunohistochemistry was performed using the SuperPicture™ 3rd Gen IHC Detection Kit (Life Technologies); sections were stained by incubation with primary antibodies and biotinylated secondary antibodies, counterstained with hematoxylin and mounted in Histomount. For immunofluorescent staining, sections were blocked with 10% donkey serum (Sigma), incubated with primary antibodies and respective secondary antibodies, counterstained with DAPI (Sigma), and mounted with CC/Mount (Sigma). Negative control for immunofluorescent staining was performed by omitting primary antibodies.
4
Oil Red O Lipid Staining Protocol
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OCT-embedded tissues were sectioned (10 µm) using a cryostat, placed onto a microscope slide, and then fixed in NBF for 15 minutes. Slides were brought to 60% isopropanol solution and then stained with freshly prepared Oil Red O solution (0.3% w/v Oil Red O in 60% isopropanol) for 15 minutes. Once stained, the slides were rinsed in 60% isopropanol solution and gently counterstained with Modified Harris Hematoxylin. Slides were rinsed with distilled water and mounted using CC mount (C9368 Sigma) and coverslip.
5
Immunohistochemical Detection of EHDV-2 in Midges
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IHC staining was performed by modification of a protocol established previously [35 (link)]. Briefly, midge sections were deparaffinized and hydrated with phosphate buffer saline (PBS). Antigens were retrieved by submerging sections in citrate-EDTA buffer (10 mM citric acid, 2 mM EDTA, 0.05% Tween®20, at pH 6.2) at 65°C for 30 min. Sections were allowed to cool at room temperature and blocked with 6% casein (Sigma-Aldrich, St. Louis, MO, USA) in PBS for 1 h. Sections were incubated at room temperature for 1 h with a 1:1,000 dilution of a polyclonal rabbit EHDV-2 primary antibody. Sections were then sequentially incubated at room temperature for 1 h with biotinylated rabbit anti-mouse secondary antibody and avidin-biotin alkaline phosphatase, according to manufacturer’s instruction (VECTASTAIN ABC-AP Staining kit, Vector Laboratories, Burlingame, CA, USA). Between each incubation step, samples were washed twice with PBST (PBS, 0.05% Tween®20) for 5 min. Sections were incubated with Vector Red chromagen substrate (Vector Laboratories) for 20 min, and counterstained with Meyer’s hematoxylin (Sigma-Aldrich) for 3 min. Sections were covered with CC Mount (Sigma-Aldrich) and air-dried. Coverslips were added using VectaMount (Vector Laboratories).
6
Immunohistochemistry Protocol for Protein Detection
7
Neuron Quantification in Rat Brains
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Quantification of the neuron numbers in the prefrontal cortex and hippocampus was conducted in the right hemisphere of 4- and 18-month-old OXYS and Wistar rats (n = 5 animals per group). Mounted sections were incubated at 60°C in a Cresyl Violet solution for 3 minutes, briefly washed in distilled H2O and dehydrated in 70%, 95%, and 100% ethanol. All slices were immersed in xylene for 2–5 min and coverslipped with CC/Mount (Sigma-Aldrich). For the estimates of neuron numbers in the middle molecular layer of the prefrontal cortex, dentate gyrus granule cell layer, and CA1 and CA3 pyramidal layer, a set of every 4–6 serial sections per animal were used. A 100× objective (Axioplan 2, Zeiss, Germany) was used to count >200 neurons per visual field. The different brain regions were defined according to the atlas developed by [43 (link)]. The dead and damaged neurons were identified by their morphology. Images of the same area of brain regions were analyzed using ImageJ (NIH, Bethesda, MD). The data in Figure 6 were presented as a percentage of the dead and damaged neurons among all neurons in each visual field of a brain region of a group.
8
Immunofluorescent Analysis of Cell Proliferation
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Day 7 MCF7–MSC co-cultures were fixed with methanol/acetone 1:1 (vol/vol) for 30 minutes at −20 °C. After fixation, cells were dried for 15 minutes and rehydrated with PBS for 15 minutes. Cells were blocked with 2 % BSA (Sigma) for 1 hour, followed by incubating with rabbit primary antibodies against Ki67 (1:200, ab15580; Abcam, Cambridge, MA, USA) or Proliferating cell nuclear antigen (PCNA) (1:100, ab2426; Abcam) in blocking solution at 4 °C overnight. After removal of primary antibodies, cells were washed three times with PBS, and then Alexa Fluor® 488 conjugated goat anti-rabbit IgG (H + L) secondary antibody (1/1000, A11008; Lifetechnologies, Carlsbad, CA, USA) was added and incubated for 2 hours at room temperature. Cells were washed three times with PBS and counterstained with 4′,6-diamidino-2-phenylindole (DAPI) nuclear dye, mounted on slides in CC/Mount (C9368; Sigma), and were observed under a Nikon®ECLIPSE Ti fluorescence microscope.
9
Picro-Sirius Red Staining for Collagen Fibers
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Picro-sirius red staining facilitates the differentiation of collagen fibers (red) from other muscle fibers and cytoplasm (yellow). Paraffin sections of eyes at E15.5 and E17.5 underwent a staining process using Picro-Sirius Red Solution (Abcam, Cambridge, MA, USA). The sections were stained for 60 minutes, followed by a rapid rinse in two changes of a 0.5% acetic acid solution. Subsequently, they were rinsed in 100% ethanol, dehydrated in two changes of 100% ethanol, and mounted with CC/Mount (Sigma-Aldrich Corp., St. Louis, MO, USA).
10
Immunodetection of Brucella in Spleen
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Formalin-fixed and paraffin-embedded tissue sections of spleen were mounted on poly-l -Lysine pre-coated slides (Sigma, USA) followed by deparaffinization in xylene, rehydration in graded ethanol, and quenching of endoperoxidase activity using 3 % H2O2 in methanol. Antigen retrieval was done in heat-mediated antigen retrieval solution according to the manufacturer’s instructions (Vector labs, USA). Then, 2.5 % normal non-immune goat serum (Vector labs) was used to block non-specific sites for 1 h at room temperature. For, both IHC and IFT, sections were incubated with rabbit anti-brucella hyper-immune serum (1 : 20) (supplied from the Division of Biological products, ICAR-IVRI) as primary antibodies. For IHC, sections were incubated with goat anti-rabbit IgG (whole molecule)-peroxidase conjugate (1 : 200) (Sigma, USA) and developed using a DAB (3,3′-diaminobenzidine tetrahydrochloride) enhanced liquid-substrate system (Sigma, USA). Immunostained sections were counter stained with Mayer’s hematoxylin (Sigma, USA) and mounted with tissue mounting medium (CC/ Mount, Sigma, USA). For IFT, goat anti-rabbit IgG (whole molecule) – FITC conjugated secondary antibodies (Sigma, USA) was used (1 : 40) followed by mounting using Fluoroshield with DAPI (4′,6-diamidino-2-phenylindole) (Sigma, USA) and viewed under fluorescent microscope (Nikon Eclipse Ti-S, Japan).
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