Cryosections of vastus lateralis muscle from the immobilized limb after 14 days of post-immobilization recovery were stained for collagen expression using picrosirius red (PSR) staining, as described previously [28 (link)]. Fifteen-micrometer sections of muscle were fixed in ice-cold acetone, washed in PBS, and stained in Bouin’s reagent (Sigma-Aldrich, St. Louis, MO, USA) for 30 min. The sections were then rinsed in water, stained with PSR (Sigma Direct Red 80, Sigma-Aldrich) for 1 h, and rinsed in acidic water (5 mL glacial acetic acid in 1 L of water) and a picric alcohol (10% picric acid, 20% ethanol) rinse. The slides were then cleared in xylene and mounted with Permount medium (Sigma-Aldrich). Slides were imaged in a blinded manner using an Olympus DP72 Digital Camera System mounted to an Olympus BX51 TRF Microscope (Olympus, Center Valley, PA, USA), and the collagen fiber area was determined using custom image analysis software (programmed by RGB; full code can be accessed at https://github.com/rbudnar/PSR ).
Dp72 digital camera system
Manufactured by Olympus
Sourced in Japan
The DP72 digital camera system is a high-performance imaging solution designed for laboratory and research applications. The DP72 features a 12-megapixel color CMOS sensor and can capture images with a wide range of resolutions and bit depths. The camera is capable of live image display and can be integrated with microscope systems for digital imaging and documentation purposes.
Automatically generated - may contain errors
Lab products found in correlation
Bx51trf microscope, by Olympus (2 mentions)
Bx43f, by Olympus (2 mentions)
Bx53 microscope, by Olympus (2 mentions)
Bouin s reagent, by Merck Group (1 mentions)
Permount medium, by Merck Group (1 mentions)
Ab79351, by Abcam (1 mentions)
Ab39973, by Abcam (1 mentions)
Ab66721, by Abcam (1 mentions)
Anti shh, by Santa Cruz Biotechnology (1 mentions)
Anti β catenin, by Santa Cruz Biotechnology (1 mentions)
Anti bmp4, by Santa Cruz Biotechnology (1 mentions)
Anti wnt10b, by Santa Cruz Biotechnology (1 mentions)
Anti gli1, by Santa Cruz Biotechnology (1 mentions)
Anti sox2, by Santa Cruz Biotechnology (1 mentions)
Toluidine blue, by Merck Group (1 mentions)
Nimp r14, by Abcam (1 mentions)
Anti dsp, by Santa Cruz Biotechnology (1 mentions)
Hematoxylin eosin he, by Merck Group (1 mentions)
Bx51 microscope, by Olympus (1 mentions)
Cellsens software, by Olympus (1 mentions)
11 protocols using dp72 digital camera system
1
Picrosirius Red Staining of Immobilized Muscle
2
Histological Analysis of Regenerated Tooth
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For the histology study, the specimens were harvested, fixed in 10% neutral buffered formalin, decalcified, and then embedded in paraffin for preparation of serial sections (5 μm thick). The sections were stained with Hematoxylin-Eosin (H&E) and examined with light microscopy.
For immunohistochemistry of regenerated tooth, briefly, after the samples were fixed with 4% PFA, decalcified, dehydrated and embedded in paraffin, they were cut into 10 μm thick sections. Serial sections were permeabilized in 0.4% Triton X-100 and blocked in PBS containing 5% BSA. Sections were incubated with primary antibodies and overnight at 4°C, then washed and incubated for 1 h at 37°C with the respective secondary antibodies followed by Hematoxylin. The primary antibodies were as follows: anti-SOX2 (ab79351, Abcam, 1:200), anti-BMP4 (ab39973, Abcam, 1:100) and anti-WNT10b (ab66721, Abcam, 1:200). Slices were analyzed using a microscope (BX43 Olympus) with an attached Olympus DP72 digital camera system.
For immunohistochemistry of regenerated tooth, briefly, after the samples were fixed with 4% PFA, decalcified, dehydrated and embedded in paraffin, they were cut into 10 μm thick sections. Serial sections were permeabilized in 0.4% Triton X-100 and blocked in PBS containing 5% BSA. Sections were incubated with primary antibodies and overnight at 4°C, then washed and incubated for 1 h at 37°C with the respective secondary antibodies followed by Hematoxylin. The primary antibodies were as follows: anti-SOX2 (ab79351, Abcam, 1:200), anti-BMP4 (ab39973, Abcam, 1:100) and anti-WNT10b (ab66721, Abcam, 1:200). Slices were analyzed using a microscope (BX43 Olympus) with an attached Olympus DP72 digital camera system.
3
Tissue Morphogenesis and Signaling Pathway Analysis
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Sections were prepared as described previously [2 (link)]. Sections were stained with haematoxylin and eosin (H&E) for the tissue morphogenetic study. Immunohistochemistry analyses were performed following the manufacturer’s instructions. The primary antibodies used were anti-Wnt10b, anti-BMP4, anti-Gli1, anti-SHH, anti-SOX2, anti-β-catenin and anti-OSR2 (Santa Cruz). Images were taken using a microscope (Olympus BX43F) with an attached Olympus DP72 digital camera system.
4
Quantifying Immunoreactive Cells in Dentate Hilus
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The DLX4+, DMRT1+, PLCB4+, and GAD67+ cells distributed in the dentate hilus were bilaterally counted in an operator-blinded manner and normalized to the number per unit area of the hilar area (Supplementary Figure 4 ). ARC+ cells distributed in the granule cell layer (GCL) were also bilaterally counted in an operator-blinded manner and normalized to the length of the SGZ (Supplementary Figure 4 ). For quantitative measurement of each immunoreactive cellular component, digital photomicrographs at 400-fold magnification were captured using a BX53 microscope (Olympus Corp., Tokyo, Japan) attached to a DP72 Digital Camera System (Olympus Corp.), and quantitative measurements were performed using the WinROOF image analysis software package (version 5.7, Mitani Corp., Fukui, Japan).
5
Mesenteric Tissue Histological Analysis
6
Immunohistochemical Analysis of Dental Tissues
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Sections were prepared as described previously [34 ]. Sections were stained with hematoxylin and eosin (H&E) for the tissue morphogenetic study. Immunohistochemistry analyses were performed following the manufacturer’s instructions. Briefly, the sections were deparaffinized and rehydrated, followed by antigen retrieval and incubation with primary antibodies at 4°C overnight. The primary antibodies used were anti-ameloblastin (1:100, Santa Cruz), anti-DSP (1:200, Santa Cruz), and anti-MMP25 (1:200, Santa Cruz). Subsequently, sections were incubated with the corresponding biotinylated secondary antibody and the avidin-biotin complex (ABC kit, Maixin, Fuzhou, China), according to the manufacturer’s protocol. Positive expressions were visualized by DAB (brown). Slides were counterstained with hematoxylin. Images were taken using a microscope (Olympus BX43F) with an attached Olympus DP72 digital camera system.
7
Histomorphometric Analysis of Chicken Jejunum
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Jejunum sections (3 cm long) were taken 2 cm away from the yolk sac. The collected sections were flushed gently using ice-cold physiological saline solution and fixed in 10% neutral buffered formalin. The middle part of the fixed sections (1 cm long) was embedded in paraffin, cross-sectioned (5 μm), placed on a glass slide, deparaffinized, and stained with Hematoxylin & Eosin (H&E, Sigma-Aldrich, St. Louis, MO). To assess villus height and crypt depth, approximately 20 fully elongated and intact crypt structures from 4 tissue slices, which were prepared from 2 different locations of the same tissue sample, were examined at 200 x magnification. Four tissue samples were randomly collected from each treatment group of chickens (n = 4). All images were captured using a BX51 microscope (Olympus America, Inc., Center Valley, PA) equipped with an Olympus DP72 Digital Camera System and analyzed using CellSens Software (Olympus, MA, CA) version 1.16.
8
Isolation and Analysis of Staged Embryonic Mandibular p4 Germs
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According to our previous research (Wang et al., 2014a (link),b (link)), the staged WZSP embryos and fetuses at E40 were obtained by cesarean section. We chose p4 germs in mandible. The p4 germs in mandibles from the same litter of staged WZSP embryos were isolated and pooled under stereo microscopy with an attached Olympus DP72 digital camera system (Olympus Corporation). The morphological stages of the p4 at E40 corresponded to the cap stage and were verified by serial histological sections as previously described (Wang et al., 2014a (link)) (Fig. S1 ).
9
Placental Immunohistochemical Analysis in GDM
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Immunohistochemistry staining was performed on sections of Formalin-Fixed and Parrffin-Embedded (FFPE) placental specimens of both maternal and fetal surfaces from GDM patients and normal pregnancies. Membranes were blocked for 1h and subsequently incubated with antibodies against TLR4, MyD88, NF-κB(p65), pIRS-1(ser312) and pAkt(ser 473) (all from Abcam, USA) overnight at 4°C and with horseradish peroxidase-conjugated secondary antibodies (Zhongshan Goldernbridge Biotechnology Co., Beijing, China) for 60 min at room temperature. Negative controls consisted of sections exposed to 2% Bovine Serum Albumin (BSA) and phosphate buffer saline (PBS) instead of primary antibody. Membranes were visualized using a 3, 3'-diaminobenzidine (DAB) kit and counterstained with hematoxylin (Zhongshan Goldernbridge Biotechnology Co., Beijing, China). Inaddition, a mouse monoclonal antibody against cytokeratin 7 (CK7) and a mouse monoclonal anti-human Vimentin (V9) antibody (from Abcam, USA) were used for identified trophoblast cellesand decidual cells, respecitively. Results were assessed based upon the evaluations of three independent observers. All slides were scanned using an Olympus IX71 microscope equipped with a DP72 digital camera system, and was analyzed with the Image-pro plus-Version 6 software.
10
Urothelial Integrity and Inflammation Assessment
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The grade of epithelial integrity and epithelial damage was assessed by the uroplakin-III (UP-III) polyclonal antibody, the E-cadherin monoclonal antibody (Cell Marque, Rocklin, CA, USA), and the zonula occludens-1 (ZO-1) polyclonal antibody (Thermo Scientific, Waltham, MA, USA), while the severity of inflammation was evaluated by the interleukin-8 (IL-8) antibody (Novus Biologicals, Centennial, CO, USA) with an immunohistochemical evaluation. Each specimen was assessed separately at ×40 magnification. The assessment was carried out using an Olympus BX51 light microscope and an Olympus DP72 digital camera system. Normal expression of UP-III, E-cadherin, and ZO-1 was defined as even distribution throughout the urothelium, mainly in the surface of the epithelial cells. Abnormal expression was defined as weak expression and distribution throughout the urothelium. Specimens with no IL-8 expression, IL-8 expression with mild vascular intensity, IL-8 expression with moderate vascular intensity, and IL-8 expression with severe vascular intensity were scored as 0, 1, 2, and 3, respectively.
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