Biopsy specimens from the cervix with LSIL, HSIL, CC, and healthy women were collected in a formalin-fixed buffer and stored at 4°C until further processing. Then, samples were embedded in paraffin, and tissue sections (5 μm) were deparaffinized and rehydrated by passage through xylene and graded ethanol solutions. Slides were treated with 3% hydrogen peroxidase containing 0.03 sodium azide in TBS (Tris buffer solution) during 5 min followed by microwave antigen retrieval at 100°C for 10 min in DAKO target retrieval solution in a H2800 Microwave processor. Next, serial sections were incubated in 0.05% albumin in TBS for 30 min at room temperature to block nonspecific protein binding. Afterwards, monoclonal antiphosphoserine antibody (Sigma Aldrich, USA), or anti-P16 antibody (Santa Cruz Biotechnology, USA), were applied to sections at 1 : 50 dilution for 60 min at 4°C. Mouse IgG Ready-To-Use was used as a negative control. DAKO EnVision+System-HRP labelled polymer anti-mouse was used as the detection system and colorized by DAB (DAKO Corporation, USA). Samples were counterstained with Mayer's modified hematoxylin (Poly Scientific, USA) before mounting and viewed under an optical microscope (Olympus BX51). Images were recorded with a DP70 digital camera (Olympus Optical Co. Ltd., Japan).
Dp70 digital camera
Manufactured by Olympus
Sourced in Japan, United States, Germany, Spain
The DP70 is a digital camera designed for microscopy applications. It features a high-resolution CCD sensor and captures digital images that can be used for documentation, analysis, and sharing. The DP70 is compatible with various Olympus microscopes and provides a direct digital imaging solution for laboratory settings.
Automatically generated - may contain errors
Lab products found in correlation
Bx51 microscope, by Olympus (56 mentions)
Bx41 microscope, by Olympus (19 mentions)
Bx61 microscope, by Olympus (10 mentions)
Bx60 microscope, by Olympus (10 mentions)
Bx51 fluorescence microscope, by Olympus (8 mentions)
Bx50 microscope, by Olympus (7 mentions)
Bx51 light microscope, by Olympus (6 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (6 mentions)
Bx51 fluorescent microscope, by Olympus (6 mentions)
Vectashield mounting medium, by Vector Laboratories (5 mentions)
Vectastain elite abc kit, by Vector Laboratories (5 mentions)
Ix71 microscope, by Olympus (4 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (4 mentions)
Ix71 inverted microscope, by Olympus (4 mentions)
Dp controller software, by Olympus (4 mentions)
Ix50 microscope, by Olympus (4 mentions)
Oil red o solution, by Cayman Chemical (3 mentions)
Bovine serum albumin (bsa), by Merck Group (3 mentions)
Triton x 100, by Merck Group (3 mentions)
Ferangi blue, by Biocare Medical (3 mentions)
299 protocols using dp70 digital camera
1
Immunohistochemical Analysis of Cervical Lesions
2
Quantifying Blood and Lymphatic Vessels in Uterine Tissues
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Each slide was examined at ×200 magnification and images captured with the use of an Olympus BX51 microscope and a DP70 digital camera with DP controller and DP manager (Olympus Optical Co., LTD, Tokyo, Japan; Figure 1(a)–(h) ). Assessments of blood and lymphatic densities were performed on 15 non-overlapping images where possible.
Blood and lymphatic vessels were counted manually for each image taken by two independent observers (N.P.N., F.M.) without knowledge of the phase of the menstrual cycle or other clinical parameters. Concordance between the two observers was calculated for the average of all fields of view.
The interclass correlation coefficients (ICCs) for inter-rater reliability were good. For polyp blood vessel density, the ICC was 0.994 (95% confidence interval (CI) 0.985–0.998; n = 20, p < 0.001), for adjacent endometrium 0.989 (95% CI 0.972–0.996; n = 8, p < 0.001), for distant endometrium 0.993 (95% CI 0.978–0.992; n = 14, p < 0.001) and for control endometrium 0.990 (95% CI 0.991–0.996, n = 32, p < 0.001).
The ICC for polyp lymphatic density was 0.991 (95% CI 0.993–0.998; n = 17, p < 0.001), for adjacent endometrium 0.986 (95% CI 0.981–0.993; n = 7, p < 0.001), for distant endometrium 0.996 (95% CI 0.987–0.995; n = 13, p < 0.001) and for control endometrium 0.993 (95% CI 0.991–0.997; n = 31, p < 0.001).
Blood and lymphatic vessels were counted manually for each image taken by two independent observers (N.P.N., F.M.) without knowledge of the phase of the menstrual cycle or other clinical parameters. Concordance between the two observers was calculated for the average of all fields of view.
The interclass correlation coefficients (ICCs) for inter-rater reliability were good. For polyp blood vessel density, the ICC was 0.994 (95% confidence interval (CI) 0.985–0.998; n = 20, p < 0.001), for adjacent endometrium 0.989 (95% CI 0.972–0.996; n = 8, p < 0.001), for distant endometrium 0.993 (95% CI 0.978–0.992; n = 14, p < 0.001) and for control endometrium 0.990 (95% CI 0.991–0.996, n = 32, p < 0.001).
The ICC for polyp lymphatic density was 0.991 (95% CI 0.993–0.998; n = 17, p < 0.001), for adjacent endometrium 0.986 (95% CI 0.981–0.993; n = 7, p < 0.001), for distant endometrium 0.996 (95% CI 0.987–0.995; n = 13, p < 0.001) and for control endometrium 0.993 (95% CI 0.991–0.997; n = 31, p < 0.001).
3
Quantitative Immunofluorescence Analysis of Complement Proteins
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Immunofluorescence was performed on OCT-embedded frozen sections after fixing with ice-cold Methanol for 10 minutes at -20°C. The sections then were incubated with 3% Goat serum in PBS-Tween (0.01%) for 1 hour at room temperature in a humidified chamber to block nonspecific antibody binding. For co-staining, the primary antibodies, mouse anti-Crry antibody (Hycult Biotech, Wayne, PA, USA) and rabbit polyclonal anti-C3 (Abcam, Cambridge, MA, USA) were applied at 1:200 dilution overnight, followed by goat anti-mouse TX-red and anti-rabbit IgG Alexa Fluor 488 (Molecular Probes, Eugene, OR, USA) for 1 hour at room temperature in a humidified chamber. Mouse and rabbit IgG (ready to use) (Dako, Glostrup, Denmark) was used as a negative control. Slides were then counterstained with Hoechst 33342 (Molecular Probes). Anti-fade mounting medium (Dako) was applied and slides were viewed and captured with a constant exposure time and aperture using a single threshold value under Olympus BX51 epifluorescence microscope, and images were recorded by a DP70 Digital Camera (Olympus Optical Co. Ltd., Tokyo, Japan). Subsequently, images were analyzed using ImageJ software and the numerical output of average intensity per nuclei was calculated.
4
Histological Analysis of Skin Samples
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Cross-sections of the full-thickness skin specimens were collected on the last day of the experiment. Skin samples were fixed for 24 h in 10% (v/v) formaldehyde and embedded in FSC 22 Frozen Section Compound (Leica Microsystems, Wetzlar, Germany). Vertical sections of 5 μm thickness were fixed onto glass slides and stained with hematoxylin and eosin (H & E) and the Masson’s trichrome stain to observe morphology and collagen formation, respectively. The slides were examined by light microscopy (Olympus BX53; Olympus Corp., Tokyo, Japan), and the images were digitally captured at a 1360 × 1024 pixel resolution with an Olympus DP70 digital camera (Olympus Corp., Tokyo, Japan).
5
Dual immunofluorescence of ZPAC and Annexin V
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Sections of specimens fixed with 4% paraformaldehyde were deparaffinized and dehydrated. After antigen activation with 10 mM
citrate buffer (pH 7.0), the sections were incubated in 50 mM Tris-HCl (pH 7.4) containing 1% BSA at RT for 1 h. Then, the
sections were incubated with both anti-ZPAC polyclonal antibody (final dilution, 1:100) and anti-annexin V (R-20) polyclonal
antibody (final dilution, 1:500; Santa Cruz Biotechnology; sc-1929) in a solution composed of 50 mM Tris-HCL and 1% BSA
overnight at 4 C. After three washes with PBS, the sections were incubated with Alexa Fluor 594-labeled donkey anti-rabbit
IgG (final dilution, 1:2,000; Invitrogen, Carlsbad, CA, USA; A-21207) for anti-ZPAC and Alexa Fluor 488-labeled donkey
anti-goat IgG antibody (final dilution, 1:2,000; Invitrogen; A-11055) for anti-annexin V in 50 mM Tris-HCl (pH 7.4)
containing 1% BSA at RT for 1 h. Subsequently, the sections were washed three times with PBS and mounted in Vectashield
mounting medium (Vector Laboratories, Burlingame, CA, USA) containing 2–5 µg/ml DAPI (Invitrogen; D1306). Finally,
fluorescence images were obtained using an Olympus BX51 microscope (Olympus) equipped with an Olympus DP70 digital camera
(Olympus). At least three independent experiments were performed for each group.
citrate buffer (pH 7.0), the sections were incubated in 50 mM Tris-HCl (pH 7.4) containing 1% BSA at RT for 1 h. Then, the
sections were incubated with both anti-ZPAC polyclonal antibody (final dilution, 1:100) and anti-annexin V (R-20) polyclonal
antibody (final dilution, 1:500; Santa Cruz Biotechnology; sc-1929) in a solution composed of 50 mM Tris-HCL and 1% BSA
overnight at 4 C. After three washes with PBS, the sections were incubated with Alexa Fluor 594-labeled donkey anti-rabbit
IgG (final dilution, 1:2,000; Invitrogen, Carlsbad, CA, USA; A-21207) for anti-ZPAC and Alexa Fluor 488-labeled donkey
anti-goat IgG antibody (final dilution, 1:2,000; Invitrogen; A-11055) for anti-annexin V in 50 mM Tris-HCl (pH 7.4)
containing 1% BSA at RT for 1 h. Subsequently, the sections were washed three times with PBS and mounted in Vectashield
mounting medium (Vector Laboratories, Burlingame, CA, USA) containing 2–5 µg/ml DAPI (Invitrogen; D1306). Finally,
fluorescence images were obtained using an Olympus BX51 microscope (Olympus) equipped with an Olympus DP70 digital camera
(Olympus). At least three independent experiments were performed for each group.
6
Immunofluorescence Analysis of SV40LT and hTERT in Transduced Chondrocytes
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Transduced articular chondrocytes were cultured in eight-well chamber slides (Millipore) to test the expression of SV40LT and hTERT. Cells were washed with phosphate-buffered saline (PBS; Dako, Agilent Technologies Spain, Spain), fixed with 4% paraformaldehyde, permeabilized with 0.5% Triton X-100, and blocked with 4% bovine serum albumin (all from Sigma-Aldrich). Subsequent incubation with two primary antibodies, mouse anti-SV40LT (SV40LT clone Pab 108; 1:100; Santa Cruz Biotechnology, USA) and rabbit anti-green fluorescent protein (GFP) labelled with Alexa Fluor 488 dye (A-21311; 1:500; Invitrogen), was performed at 4°C overnight.
After incubation with primary antibodies, cells were washed three times with PBS and incubated with a goat anti-mouse secondary antibody labelled with Alexa Fluor 594 dye (A-11032; 1:1,000; Thermo Fisher Scientific) at room temperature for one hour. After three additional washes in PBS, a two-minute incubation with Hoechst (bisBenzimide H 33342 trihydrochloride, Sigma-Aldrich) was performed. Slides were mounted with Glycergel aqueous mounting medium (Dako) and observed using an Olympus BX61 fluorescence microscope (Olympus Iberia, Spain) coupled to an Olympus DP70 digital camera (Olympus Iberia). Fluorescence micrographs were obtained employing the cellSens Dimension software (Olympus Iberia).
After incubation with primary antibodies, cells were washed three times with PBS and incubated with a goat anti-mouse secondary antibody labelled with Alexa Fluor 594 dye (A-11032; 1:1,000; Thermo Fisher Scientific) at room temperature for one hour. After three additional washes in PBS, a two-minute incubation with Hoechst (bisBenzimide H 33342 trihydrochloride, Sigma-Aldrich) was performed. Slides were mounted with Glycergel aqueous mounting medium (Dako) and observed using an Olympus BX61 fluorescence microscope (Olympus Iberia, Spain) coupled to an Olympus DP70 digital camera (Olympus Iberia). Fluorescence micrographs were obtained employing the cellSens Dimension software (Olympus Iberia).
7
Direct Cell Counting in Sludge Samples
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Regarding cell counting, 5 mL of a mixed-liquor sludge sample from ANSBRs was added to a BD Falcon™ tube, sonicated for 100 s (20 kHz; output power 50 W), and diluted with filter-sterilized phosphate-buffered saline (pH 7.0). Aliquots (10–50 μL) of these diluted samples were taken and used for direct cell counting. The direct total count was measured by epifluorescence microscopy with 4,6-diamidino-2-phenylindole (DAPI) or SYBR Green I (Invitrogen Corporation, Carlsbad, CA, USA) staining as described (22 (link), 76 (link)). A direct viable count was also obtained using a Molecular Probe LIVE/DEAD®BacLight™ Viability kit (Invitrogen) as described (24 (link), 81 ). Stained specimens were observed under an Olympus model BX-50 phase-contrast/epifluorescence microscope equipped with an Olympus DP70 digital camera (Olympus Corporation, Tokyo, Japan).
8
Rice Leaf Semithin Sectioning and Imaging
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Semi-thin sectioning was performed as described previously64 (link)65 (link). The fifth leaves of 6-week-old rice were fixed in 2.5% of glutaraldehyde and post-fixed with 1% of OsO4 at room temperature. Samples were dehydrated through a graded acetone series (10% increments) for 60 minutes each. Infiltrated was started with 20% of Spurr’s resin and then 40%, 60%, and 80% of Spurr’s resin every 3 hours. Following infiltration in three changes of 100% Spurr’s resin for 24 hours each, samples were finally embedded in 100% Spurr’s resin and polymerized at 60 °C overnight. Semi-thin sections (0.5 μm) were made using an RMC MT-7 ultramicrotome (Reichert-Jung, Depew, NY, USA) and were stained with 0.25% Toluidine Blue O. Images were photographed by an Olympus BX51 microscope equipped with an Olympus DP 70 digital camera (Olympus, Center Valley, PA, USA).
9
Fluoro-Jade C Staining of Degenerating Axons
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The slides were deparaffinized in xylene, rehydrated in ethanol and water, and then treated for 10 min with a 0.06% potassium permanganate solution. Sections were rinsed twice with distilled water (dH2O) for 1 min and incubated in 0.0001% Fluoro-Jade C (Chemicon, Millipore, Billerica, MA, USA) staining solution for 20 min in the dark. After that, they were washed in dH2O thrice per minute and dried on a hot plate on 50 °C for 20 min. Sections were dehydrated in xylene two times for 10 min, mounted in Entellan® (Merck Millipore, Billerica, MA, USA), and coverslipped. Stained sections were examined by epifluorescence microscopy using the appropriate light filter cube (Olympus BX 51 microscope with Olympus DP 70 digital camera, Olympus, Tokyo, Japan).
Quantification of Fluoro-Jade C intensity in the OT was done on microphotographs taken at ×400 final magnification; for each animal, two images were used for the analyses. Within each microphotograph, three ROIs of 0.0057 mm2 were analyzed. By subtracting the background fluorescent intensity from those ROIs, we could determine only degenerating axons within that field.
Quantification of Fluoro-Jade C intensity in the OT was done on microphotographs taken at ×400 final magnification; for each animal, two images were used for the analyses. Within each microphotograph, three ROIs of 0.0057 mm2 were analyzed. By subtracting the background fluorescent intensity from those ROIs, we could determine only degenerating axons within that field.
10
Histological Analysis of Liver and Aorta
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After 8 weeks of treatments, the liver tissues and aorta were collected, fixed in 10% neutral buffered (formaldehyde), and sliced (5 μm thick) and trimmed transversely into serial sections using a cryo-microtome (Leica Biosystems, Richmond, VA, USA). Sections were later deparaffinized in xylene, rehydrated through an ascending ethanol series, and stained with hematoxylin-eosin (H&E). Oil Red-O staining was used to analyze fat deposition in liver tissues. The liver tissues were embedded in FSC 22 frozen section compound (Leica Biosystems, Richmond, VA, USA) and sectioned (5 μm thickness) using a cryostat (Tissue-Tek® Cryo3® Flex Microtome/Cryostat, Sakura, Leiden, The Netherlands). The sections were stained with Oil Red-O staining, and mounting was performed with xylene-based media. The slides were observed under an Olympus BX 61 microscope (Olympus, Tokyo, Japan) at 200× magnification and images were acquired using an Olympus DP70 digital camera (Olympus).
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