Immunostaining of laminin was performed as described previously [27 (link)]. For immunofluorescent staining of PDCD10, sections were incubated with rabbit anti-PDCD10 (Atlas, 1:65) at 4 °C overnight and then incubated with biotinylated goat anti-rabbit IgG (Dako, 1:400) at 37 °C for 1 h followed by the substrate reaction with FITC-labelled avidin (Dako, 1:400) at room temperature for 1 h. For double-immunofluorecent staining, the following antibody mixtures were applied to the sections: rabbit anti-PDCD10 (Atlas, 1:65) and mouse anti-GFAP (Sigma, 1:200); rabbit anti-PDCD10 and mouse anti-CD31 (Dako, 1:20); rabbit anti-PDCD10 and mouse anti-CD68 (Dako, 1:100); rabbit anti-PDCD10 and mouse anti-PCNA (Dako, 1:200); mouse anti-PDCD10 (Santa Cruze, 1:50) and rabbit anti-caspase 3 (active form) (Cell signaling, 1:400). After incubation overnight, the sections were incubated with the mixture of biotinylated goat anti-rabbit IgG (1:400) and Texas red anti-mouse IgG (H + L) (Vector Laboratories, 1:200) followed by the substrate reaction with FITC-labelled avidin. Counterstaining was performed with Hoechst-33258. The sections were finally analyzed by using a fluorescence microscope with ApoTome System (Zeiss, Axio Imager M2).
Mouse anti pcna
Manufactured by Agilent Technologies
Sourced in United States
Mouse anti-PCNA is a laboratory reagent that detects the Proliferating Cell Nuclear Antigen (PCNA) protein. PCNA is a key component of the DNA replication machinery and is commonly used as a marker for cell proliferation.
Automatically generated - may contain errors
Lab products found in correlation
Axio imager m2, by Zeiss (1 mentions)
Mouse anti gfap, by Merck Group (1 mentions)
Rabbit anti caspase 3, by Cell Signaling Technology (1 mentions)
Apotome system, by Zeiss (1 mentions)
Mouse anti cd31, by Agilent Technologies (1 mentions)
Biotinylated goat anti rabbit igg, by Agilent Technologies (1 mentions)
Mouse anti cd68, by Agilent Technologies (1 mentions)
Biotinylated goat anti rabbit igg, by Vector Laboratories (1 mentions)
Rabbit anti pdcd10, by Agilent Technologies (1 mentions)
Texas red anti mouse igg h l, by Vector Laboratories (1 mentions)
Triton x 100, by Merck Group (1 mentions)
Pbs 1x, by Thermo Fisher Scientific (1 mentions)
Citrate buffer, by Zytomed Systems (1 mentions)
H 3403, by Vector Laboratories (1 mentions)
Meyer s hematoxylin, by Merck Group (1 mentions)
Dako envision kit, by Agilent Technologies (1 mentions)
Ab6308, by Abcam (1 mentions)
Ab9758, by Abcam (1 mentions)
Mouse anti fibronectin, by Santa Cruz Biotechnology (1 mentions)
Diaminobenzidine hydrogen peroxidase, by Agilent Technologies (1 mentions)
5 protocols using mouse anti pcna
1
Immunostaining of PDCD10 in Tissue Sections
2
PCNA Immunohistochemistry in Tissue Sections
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For immunohistochemistry (IHC), sections were deparaffinized and hydrated. Serial slides were used for PCNA (Proliferating Cell Nuclear Antigen) staining. Tissue sections were treated with 0.1% triton X-100 (Sigma-Aldrich) in PBS 1x (Life Technologies) at room temperature (RT) for 10 min. Then endogenous peroxidases were inhibited by incubation with 3%H2O2 in methanol at RT for 10 min. Tissue sections were then placed in an antigen retrieval solution (0.01 M citrate buffer, pH = 6 (Zytomed, Berlin, Germany) for 15 min at 350 W) and quenched for endogenous peroxidases as described above. After saturation (GBI labs, Washington, USA), mouse anti-PCNA (M0879, DakoCytomation, Courtaboeuf, France) at 525 µg/ml was applied for 1 h at 37 °C. Sections were incubated with a kit anti-mouse HRP (GBI labs) for 30 min at RT. Staining was developed with HRP Green (Zytomed). Then, sections were counterstained with nuclear fast red (H3403, VectorLabs, Burlingame, CA, USA), dehydrated and mounted. Isotype control antibodies are used as negative controls.
3
Quantitative Analysis of Tumor Proliferation
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Representative tumor sections for each group were stained with H&E and then examined using a light microscope (Thornwood, NY, Carl Zeiss Inc.). Additionally, the tumor tissue sections were incubated at 4 °C overnight with mouse anti-PCNA (DAKO, Glostrup, Denmark) to detect proliferating tumor cell population via immunohistochemistry. The slides were counterstained with Meyer’s hematoxylin (St. Louis, MO, Sigma). The positive spots of PCNA were semiquantitatively assessed using the IHC Profiler Plugin for ImageJ software (National Institutes of Health, Bethesda, MD, USA).
4
Keloid Spheroid Immunohistochemistry Analysis
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Representative sections were stained with hematoxylin and eosin (H & E) and Masson’s trichrome, and then examined by light microscopy. Keloid spheroid sections were incubated at 4 °C overnight with mouse anti-collagen type I (ab6308; Abcam, Ltd., Cambridge, UK), mouse anti-collagen type III (C7805; Sigma, St. Louis, MO), mouse anti-elastin (E4013; Sigma), mouse anti-fibronectin (sc-52331; Santa Cruz Biotechnology), rabbit anti-TGF-β1 (ab9758; Abcam, Cambridge, UK), mouse anti-EGFR (Ab-1; Oncogene Research Products, Calbiochem), rabbit anti-Erk 1/2 (#4370 S; Cell Signaling Technology, Beverly, MA), and rabbit anti-Smad 2/3 complex (#8685 S; Cell Signaling Technology) mouse anti-PCNA (DAKO), goat anti-Cytochrome c (SC-8385; Santa Cruz Biotechnology) primary antibody, and then incubated at room temperature for 20 min with the Dako Envision™ Kit (DAKO, Glostrup, Denmark) as secondary antibody. Diaminobenzidine/hydrogen peroxidase (DAKO, Carpinteria, CA) was used as the chromogen substrate. All slides were counterstained with Meyer’s hematoxylin. The expression levels of TGF-β1, EGFR, Erk 1/2, Smad 2/3, type I and III collagen, elastin, and fibronectin were semi-quantitatively analyzed using MetaMorph® image analysis software (Universal Image Corp., Buckinghamshire, UK). Results are expressed as the mean optical density for six different digital images.
5
Immunofluorescence Staining of Tissue Sections
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Immunofluorescence staining was performed as described previously [24 (link)]. Briefly, 50-μm-thick vibratome or 4-μm-thick wax-embedded sections or MDCK cells were blocked and incubated with one or more primary antibodies. The following primary antibodies were used: rabbit anti-Prox1 (Millipore; Billerica, MA, USA), rabbit anti-LYVE-1 (Research Diagnostics; Flanders, NJ, USA), rabbit anti-NKCC2 (courtesy of Dr. Mark A. Knepper, NIH), goat anti-aldose reductase (AR; courtesy of Dr. Peter Kador, NIH), rabbit anti-AQP2 (Millipore), rabbit anti-CLC-K (Millipore), rabbit anti-AQP1 (Millipore), rat anti-CD31 (BD Bioscience; Franklin Lakes, NJ, USA), goat anti-THP (Mpbiomedicals; Aurora, OH, USA), mouse anti-PCNA (Dako; Carpinteria, CA, USA), and rabbit anti-TonEBP. Appropriate Alexa 488- (Invitrogen; Carlsbad, CA, USA), Cy3-, or Cy5- (Jackson ImmunoResearch Laboratories; West Grove, PA, USA) conjugated secondary antibodies were used for fluorescent detection; nuclei were stained with 4',6-diamidino-2-phenylindole (DAPI; Roche Molecular Biochemicals; Indianapolis, IN, USA). For wax-embedded sections, citrate buffer (pH 6.0) was used for antigen retrieval.
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