The status of GSTP expression and the extent of cell proliferation in the livers of control and experimental rats were assessed by a double immunostaining technique. Briefly, formalin-fixed paraffin-embedded liver sections were deparaffinized, rehydrated, and immunostained with anti-proliferating cell nuclear antigen (PCNA) antibody (1:200; Dako, Glostrup, Denmark) followed by immunostaining for anti-GSTP antibody (1:500, Medical and Biological Laboratories, Tokyo, Japan), as previously described.9 (link)Immature vessels and/or vessels undergoing angiogenesis were detected by CD34 staining.16 (link) Liver sections were deparaffinized, rehydrated, and immunostained with anti-CD34 antibody (1:100; R&D Systems, Minneapolis, USA). All CD34 positive vessels in each liver section were counted. All sections were examined by light microscopy (Axio Imager 2, Carl Zeiss, Jena, Germany); AxioVision LE 4.8.2.0 digital image processing software (Carl Zeiss) was used for quantification. The image analysis was conducted by pathologists blinded to the treatments. All immunohistochemical analyses were conducted in duplicate and the experiments were repeated twice.
Pcna antibody
Manufactured by Agilent Technologies
Sourced in Denmark, United States
The PCNA (Proliferating Cell Nuclear Antigen) antibody is a laboratory reagent used to detect the presence and localization of PCNA, a protein involved in DNA replication and repair processes. The PCNA antibody can be used in various analytical techniques, such as immunohistochemistry, Western blotting, and immunofluorescence, to study cellular proliferation and DNA damage response mechanisms.
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Lab products found in correlation
Stereo lumar v12 stereomicroscope, by Zeiss (2 mentions)
Tcs lsi zoom confocal microscope, by Leica (2 mentions)
Anti cd34 antibody, by R&D Systems (1 mentions)
Axio imager 2, by Zeiss (1 mentions)
3 3 diaminobenzidine dab substrate kit, by Vector Laboratories (1 mentions)
Varistain 24 4 automatic slide stainer, by Thermo Fisher Scientific (1 mentions)
Hv f202scl camera, by Hitachi (1 mentions)
Hematoxylin, by Vector Laboratories (1 mentions)
Apoptosis detection kit, by Merck Group (1 mentions)
Avidin biotin peroxidase complex abc method, by Vector Laboratories (1 mentions)
3 3 diaminobenzidine (dab), by Vector Laboratories (1 mentions)
Histofine simple stain max po, by Nichirei Biosciences (1 mentions)
Axio imager 2 light microscope, by Zeiss (1 mentions)
Apotome 2 system, by Zeiss (1 mentions)
Alexa fluor 488 conjugated secondary antibody, by Thermo Fisher Scientific (1 mentions)
Anti cleaved caspase 3 antibody, by Cell Signaling Technology (1 mentions)
6 protocols using pcna antibody
1
Liver Cell Proliferation and GSTP Assessment
2
Histological Imaging of Tadpole and Froglet Tissues
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Living and dissected tadpoles and froglets were imaged on a Carl Zeiss StereoLUMAR.V12 stereomicroscope. Tissues and tumors were fixed in 4% PFA (paraformaldehyde), dehydrated, and embedded in paraffin. Sections of 5 μm were rehydrated and stained with hematoxylin and eosin for histological examination. For PCNA immunohistochemistry, 5-μm sections were pressure cooked in citrate buffer (10 mM citric acid, 0.05% tween-20, pH 6) for antigen retrieval and blocked in 3% goat serum, 1% BSA, 0.1% tween-20 in PBS. Slides were incubated overnight with PCNA antibody (Clone PC10-Dako) at 4°C. Secondary goat anti-mouse dylight-488 conjugated antibody was used for detection. Fluorescent tumor sections were imaged on a Leica TCS LSI zoom confocal microscope.
3
Immunohistochemical Analysis of Liver Lesions
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Liver slides were incubated overnight at 4°C with a polyclonal rabbit anti-rat glutathione S-transferase placental form (GST-P) antibody (1 : 500 dilution, Medical & Biological Laboratories Co., Ltd., Japan) and proliferating cell nuclear antigen (PCNA) antibody (1 : 500, Dako, USA). Bound antibodies were visualized by 3,3′-diaminobenzidine (DAB) substrate kit (Vector Laboratories, USA), and nuclei were counterstained using Mayer's hematoxylin. The number and area (mm2) of GST-P-positive foci were analyzed per liver section (cm2) as previously described [38 (link)], while the PCNA-positive cells (brown color nuclei) were quantified using ImageJ software version 1.44.
4
Histological Analysis of Tadpole Development
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All pictures of euthanized and living tadpoles were taken with a Carl Zeiss StereoLUMAR.V12 stereomicroscope. Complete heads of euthanized tadpoles were dissected, fixed in 4% PFA (paraformaldehyde) and decalcified by Morse’s solution (10% sodium citrate and 22.5% formic acid) for 7 hours up to overnight (depending on age of specimen). Tissue samples were subsequently dehydrated and embedded in paraffin. Tissue sections of 5 μM were cut by microtome, rehydrated and hematoxylin and eosin stained with a Varistain™ 24-4 Automatic Slide Stainer (Thermo-Scientific). Images were captured with an Axio Scan.Z1 (Zeiss, Germany). Images were acquired with a 20X Plan-Apochromat 0.8 NA dry objective, using a Hitachi HV-F202SCL camera. For PCNA immunohistochemistry, antigen retrieval was performed using the PickCell 2100-Retriever (ProteoGenix) in citrate buffer (10 mM citric acid, 0.1% Tween-20, pH 6). Slides were subsequently blocked with blocking buffer (3% goat serum, 1% BSA, 0.1% Tween-20) and incubated overnight with PCNA antibody (Clone PC10-Dako) diluted 1/500 in blocking buffer at 4 °C. Detection was performed with secondary goat anti-mouse dylight-594 and counter-staining was performed with Hoechst-33342. Sections were subsequently imaged using a Leica TCS LSI zoom confocal microscope.
5
Histological and Immunohistochemical Analysis of Tumor Samples
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The animals were euthanized on day 15, and the tumors were removed carefully and fixed in 10% formalin over 24 h. The tissues were then dehydrated in an alcohol–xylene series and embedded in paraffin wax. From each block, 2-μm-thick sections were prepared and stained with hematoxylin (Vector, Burlingame, CA, USA) and eosin (H&E) for histological examination. For immunohistochemistry, the sections were incubated in 3% H2O2 in methanol for 10 min to remove endogenous peroxidase and blocked with 1% BSA in PBS for 1 h. The sections were then incubated with PCNA antibody (Dako, Carpinteria, CA, USA) overnight at 4°C. After washing three times with PBS-T, the sections were subjected to the avidin–biotin peroxidase complex (ABC) method (Vector), and peroxidase activity was evaluated with 3,3′-diaminobenzidine (Vector). Finally, the sections were counterstained with hematoxylin. The terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assay was done using an Apoptosis Detection Kit (Millipore, Billerica, MA, USA) according to the manufacturer’s protocol. PCNA-positive cells and apoptotic cells were counted from five randomly selected areas under 200× magnification and represented as mean ± SD.
6
Histological and Molecular Characterization of Tissue Samples
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Histological examinations were performed in hematoxylin and eosin (H&E)‐stained sections. Cell proliferation was analyzed using a mouse monoclonal anti‐proliferating cell nuclear antigen (PCNA) antibody (M0879; Dako, Glostrup, Denmark). Cell apoptosis was evaluated using anti‐cleaved caspase 3 antibody [#9664; Cell Signaling Technology (CST), Danvers, MA, USA]. Immunostaining for PCNA and cleaved caspase 3 was detected using Histofine® Simple Stain MAX PO (Nichirei, Tokyo, Japan). Immunofluorescence analyses were carried out using anti‐VEGF primary antibody (19003‐1‐AP; Proteintech, Rosemont, IL, USA), anti‐PDGFRβ primary antibody (#3169; CST), and Alexa Fluor 488‐conjugated secondary antibody (A32731; Invitrogen, Carlsbad, CA, USA). Images were analyzed with an Axio Imager 2 light microscope and ApoTome.2 system (Carl Zeiss, Oberkochen, Germany).
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