Mice were anesthetized with isoflurane and perfused with PBS followed by 4% PFA at 11 am for SD and SD-Cont, and at 2 pm for RS and RS-Cont. Brains were fixed with 4% PFA overnight and placed into 30% sucrose until saturated. Thirty-micrometer cryosections were collected into PBS and stored in cryoprotectant at −20°C. For immunofluorescent staining, samples were stained using primary antibodies: anti-Nkx2-1 (TTF-1) (1:500, ab76013; Abcam) and secondary antibodies. To stain cFos, samples were stained with anti-cFos (1:1,000, 226003; Synaptic Systems) and universal biotinylated anti-mouse/rabbit IgG (Universal Elite ABC kit, PK-7200; Vector laboratories) antibodies with Universal Elite ABC kit and developed with Vector SG substrate kit peroxidase (SK-4700: Vector laboratories). The number of cFos-positive cells was quantified by visual scoring. Genotypes and conditions were blinded during the experimental procedures. For MAP2 immunofluorescence, 25-μm cryosections were stained using anti-MAP2 (1:100, ab5392; Abcam).
Universal elite abc kit
Manufactured by Vector Laboratories
Sourced in United States, United Kingdom
The Universal Elite ABC kit is a versatile and reliable reagent system designed for immunohistochemical staining. It provides a sensitive detection method for a wide range of primary antibodies in various tissue types. The kit contains the necessary components to perform the Avidin-Biotin Complex (ABC) detection method, a well-established technique in immunohistochemistry.
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Lab products found in correlation
Diaminobenzidine tetrahydrochloride solution kit hk153 5k, by BioGenex (2 mentions)
Anti h3k27me3, by Cell Signaling Technology (1 mentions)
Anti pcreb, by Cell Signaling Technology (1 mentions)
Ki67 antibody clone mib 1, by Agilent Technologies (1 mentions)
Vector r t u vectastain kit, by Vector Laboratories (1 mentions)
Ab5392, by Abcam (1 mentions)
Ab76013, by Abcam (1 mentions)
Ab32199, by Abcam (1 mentions)
A4502, by Agilent Technologies (1 mentions)
Ab175430, by Abcam (1 mentions)
Ab203829, by Abcam (1 mentions)
Abc ap mouse igg kit, by Vector Laboratories (1 mentions)
Bx53 dp74 microscope, by Olympus (1 mentions)
Immpact dab, by Vector Laboratories (1 mentions)
Image pro plus software version 6, by Media Cybernetics (1 mentions)
Epifluorescence microscope, by Leica (1 mentions)
Immuno mount, by Thermo Fisher Scientific (1 mentions)
Hoechst 33342, by Thermo Fisher Scientific (1 mentions)
12 protocols using universal elite abc kit
1
Immunofluorescent Staining of Nkx2-1 and cFos
2
Immunohistochemical Detection of p16 Protein
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The procedure followed the method as described previously 12 (link). Briefly, for immunohistochemical (IHC) staining, cultured cells were grown on SlideFlasks with bottom detachable slides (Nalge Nunc, Naperville, IL) that could be used for IHC staining directly later. The samples (slides) were first incubated with 1% H2O2 for 30 min. The samples were incubated with first antibody against human p16 (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) for 16 h at 40C, then by corresponding second antibody and the Universal Elite ABC Kit (Vector Laboratories, Inc., Burlingame, CA) according to the manufacturer's protocol. The reaction was visualized with DAB solution (75 mg 3,3'-Diaminobenzidine and 30 ml 50% H2O2 in 150 ml PBS) for 3-10 min.
3
E-cadherin Immunohistochemistry Staining Protocol
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Immunohistochemistry was performed using tissue microarray sections. Four-micrometer-thick sections were transferred to poly-l-lysine-coated glass slides and incubated in a dry oven at 60°C for 1 h. The sections were then dewaxed using xylene (3 changes), rehydrated in a descending series of graded ethanol concentrations, and rinsed using Tris-buffered saline (pH 7.4). The endogenous peroxidase activity was blocked using 5% hydrogen peroxide in methanol for 15 min at 37°C. For antigen retrieval, the slides were placed in citrate buffer (10% citrate buffer stock in distilled water, pH 6.0) and microwaved for 10 min. Non-specific staining was blocked using 1% horse serum in Tris-buffered saline (pH 7.4) for 3 min. The primary antibody for E-cadherin was diluted 1:100 (BD Biosciences, San Jose, CA), and the immunostaining was developed using an avidin-biotin-peroxidase complex (Universal Elite ABC Kit; PK-6200; Vectastain, Burlingame, CA, USA) and diaminobenzidine tetrahydrochloride solution (HK153-5K; Biogenex, San Ramon, CA, USA). Positive controls (samples with known reactivity for the antibody) and negative controls (omission of the primary antibody) were included in each assay. Positive E-cadherin staining was defined as a cytoplasmic membranous pattern, and was classified as high (>90%), low (0–90%), or absent (0%) [78 (link)].
4
Immunohistochemical Profiling of Prostate Samples
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TMA containing 78 cases of formalin fixed and paraffin embedded normal or cancer prostate samples were obtained through clinical protocols approved by the Institutional Review Board of Baylor College of Medicine. We have complied with all relevant ethical regulations. The TMAs were dewaxed in 60 °C oven for 2 h and deparaffinized, and rehydrated through incubating in xylene and alcohol series. Tissue sections were subjected to antigen retrieval in 10 mM sodium citrate buffer (pH 6.0) in a food steamer for 45 min. The Universal Elite ABC kit (Vector Laboratories) was used for immunohistochemistry (IHC) staining, according to the manufacturer’s instruction. After suppressing endogenous peroxidase activity, the sections were incubated in normal horse serum to prevent nonspecific immunoglobulin binding, then incubated with primary antibody overnight at 4 °C. Primary antibodies used were anti-p-CREB and anti-H3K27me3 from Cell Signaling Technology. A streptavidin-HRP detection system was used to reveal specific binding. The stained slides were scored by two investigators who reached consensus, as following: staining intensity −/+, <25% positive cells (weak, score 1); staining intensity ++, 25–50% positive cells (intermediate, score 2); and staining intensity +++, >50% positive cells (strong, score 3).
5
Immunohistochemical Analysis of p-CREB and GRK3
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The Universal Elite ABC kit (Vector Labs) was used for immunohistochemistry (IHC) staining, according the manufacturer's instructions. Briefly, slides of five micrometer sections from 78 cases of formalin-fixed and paraffin-embedded prostate cancer and normal tissue blocks were dewaxed in 60°C oven for 2 hours and rehydrated through incubating in xylene and alcohol series. Antigen retrieval was done in 10 mmol/L sodium citrate buffer (pH 6.0) in a food steamer for 30 minutes. After suppressing the endogenous peroxidase activity the sections were incubated in normal horse serum to prevent nonspecific immunoglobulin binding. Upon PBS wash, the sections were then treated with the anti-human p-CREB (Cell signaling, USA) or anti-human GRK3 antibody (Epitomics, USA) at 4°C overnight. A streptavidin-HRP detection system was used to reveal specific binding. Immunoreactivity was scored as following: staining intensity −/+, <25% positive cells (weak, score 1); staining intensity ++, 25–50% positive cells (intermediate, score 2); and staining intensity +++, >50% positive cells (strong, score 3). Percent of positive cells and staining intensity were scored independently by two experienced researchers.
6
Immunohistochemical Analysis of Tumor Proliferation
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Formalin-fixed, paraffin-embedded, 4-µm tumor sections were dewaxed in xylene, rehydrated with graded alcohol concentrations, and placed in an endogenous peroxide blocking buffer for 15 minutes17 (link). Sections were washed in water, antigen-retrieved, and placed in citrate buffer17 (link). Nonreactive staining was blocked by treating the sections with 1% horse serum in Tris-buffered saline (pH 6.0) for 3 minutes17 (link). Ki-67 antibody (clone MIB1, IS626, 1:200; DAKO, Denmark) was then applied and the binding of antibodies was detected using the avidin-biotin-peroxidase complex (Universal Elite ABC Kit; Vectastain, Burlingame, CA, USA) for 10 minutes. Diaminobenzidine tetrahydrochloride solution (Kit HK153–5K; Biogenex, San Ramon, CA, USA) was used as a chromogen17 (link). The tumor specimens were stained with hematoxylin and eosin (H&E) for the examination of the basic histomorphological features17 (link).
7
Immunohistochemical Analysis of Tumor Tissue
8
Immunohistochemical Analysis of KIT and PTEN
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Formalin-fixed, paraffin-embedded, 4-μm tumor sections were dewaxed in xylene, rehydrated with graded alcohol concentrations, and placed in an endogenous peroxide blocking buffer for 15 minutes. Sections were washed in water, antigen-retrieved, and then placed in citrate buffer. Nonreactive staining was blocked by treating the sections with 1% horse serum in Tris-buffered saline (pH 6.0) for 3 minutes. Then, anti-KIT (1:800, A4502, DAKO, Carpinteria, CA, USA) and anti-phosphatase and tensin homolog (PTEN, 1:100, ab32199; Abcam, Cambridge, MA, USA) antibodies were then applied and the antibody binding was detected using an avidin-biotin peroxidase complex (Universal Elite ABC kit, Vectastain, Burlingame, CA, USA) for 10 minutes. Diaminobenzidine tetrahydrochloride solution (Kit HK153-5K; Biogenex, San Ramon, CA, USA) was then used as a chromogen. The tumors specimens were stained with hematoxylin and eosin (H&E) to examine the basic histomorphological features.
9
Immunohistochemical Analysis of EMT Markers
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The HE staining protocol was performed according to the standard laboratory protocol. Immunohistochemical expression analyses were performed on 3-μm-thick FFPE tissue sections using the following antibodies: anti-human vimentin mouse monoclonal (1:50, clone: V9; #ab8069; Abcam, Cambridge, UK), anti-human E-cadherin mouse monoclonal (1:50, clone: 36/E-Cadherin; BD Bioscience, NJ), anti-human ZEB1 rabbit monoclonal (1:50, clone:EPR17375;ab203829; Abcam, Cambridge, UK), anti-human Twist mouse monoclonal (1:50, clone:10E4E6;ab175430; Abcam, Cambridge, UK), Universal Elite ABC kit (PK-6200; Vector Laboratories, Burlingame, CA), Elite ABC Mouse kit (PK-6102; Vector Laboratories, Burlingame, CA), and ABC-AP Mouse IgG kit (AK-5002; Vector Laboratories). Tissue sections were incubated in ImmPACT DAB (Vector Laboratories) until the desired staining intensity developed. The sections were then counterstained with hematoxylin and mounted. Stained sections were viewed under an Olympus BX53+DP74 microscope.
10
Immunohistochemical Analysis of Tumor Samples
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After the termination of the animal experiments, the excised tumors were fixed in 10% neutral-buffered formalin and embedded in paraffin blocks. The blocks were then cut to produce 5-µm thick tissue sections. The sections were stained with either H&E alone, or with antibodies against α7nAChR (1:50 dilution) or vimentin (1:200 dilution). For immunohistochemical studies, the sections were rehydrated with PBS and processed as previously described (6 (link)). Sections were rinsed in dH2O and antigens were retrieved by microwaving. After the sections were cooled and rinsed three times in dH2O and twice in PBS, staining was performed according to the manufacturers protocol (Universal Elite ABC kit; Vector laboratories, Inc., Burlingame, CA, USA). All stained slides were visualized with a Leica DMI 3000B microscope 2005 (Leica, Wetzlar, Germany). Tumor sections were scanned at a magnification of ×10, and representative images at a magnification of ×20 are presented. The relative quantities of protein in the positively-stained regions were quantified for the integrated optical density using Image-Pro Plus software, version 6.0 (Media Cybernetics, Inc., Rockville, MD, USA).
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