A set of full-thickness skin samples were obtained from post-mortem tissue harvesting, fixed overnight in 4% paraformaldehyde, and then dehydrated for paraffin embedding and slide generation; hematoxylin and eosin staining according to standard protocol. For immunohistochemistry, formalin-fixed, paraffin-embedded tissue slides were cleared and rehydrated through a series of ethanol washes. Antigen retrieval (20 min in pressure cooker, microwaved at 100 power) was performed with 10mM citrate. Slides were incubated in hydrogen peroxide (30 min at 4 degrees C) and then blocked with 10% goat serum/0.1% tween for 1 hour at room temperature. Primary antibodies were added and incubated overnight. The following antibodies were used: rabbit Sox9 1:800 (Abcam 185667), rabbit Ki67 1:200 (Abcam 16667), phosphor-EGFR 1:500 (Abcam 40815), IL6 1:500 (Abcam 6672), and CD11b 1:250 (BD Pharmingen 550282). Sections were washed the following day with 0.1% PBST and incubated with rabbit secondary HRP-labeled polymer (Dako) for 1 hour at room temperature, and then quickly washed with 0.1% PBST and PBS. AEC chromogen (Dako) was used for the colometric development reaction. Slides were then briefly counterstained with hemotoxylin, mounted with Faramount Aqueous Mounting Media (Dako) and sealed for subsequent visualization by light microscopy.
Aec chromogen
Manufactured by Agilent Technologies
Sourced in United States
The AEC chromogen is a laboratory equipment product designed for use in immunohistochemistry applications. It functions as a chromogenic substrate, enabling the visualization of target antigens in tissue samples. The AEC chromogen produces a red-brown color reaction when exposed to the enzyme label in an immunohistochemical staining procedure.
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Lab products found in correlation
Control igg, by Santa Cruz Biotechnology (2 mentions)
Mmp 9, by Santa Cruz Biotechnology (2 mentions)
Human cat no br1008, by Tissue Array (2 mentions)
Kdm3a, by Fortis Life Sciences (2 mentions)
Hematoxylin, by Merck Group (2 mentions)
Faramount aqueous mounting media, by Agilent Technologies (1 mentions)
Rabbit ki67, by Abcam (1 mentions)
Interleukin 6 (il 6), by Abcam (1 mentions)
Cd11b, by BD (1 mentions)
Mirax viewer software, by Zeiss (1 mentions)
Ab214468, by Abcam (1 mentions)
Axiocam mrc camera, by Zeiss (1 mentions)
Envision flex target retrieval solution high ph, by Agilent Technologies (1 mentions)
Eosin y, by Carl Roth (1 mentions)
Cd68 ab125212, by Abcam (1 mentions)
Cd3 ab5690, by Abcam (1 mentions)
Biotinylated anti rabbit secondary antibody, by Vector Laboratories (1 mentions)
Aquatex, by Merck Group (1 mentions)
Antibody diluent, by Biocare Medical (1 mentions)
Anti cd44, by BD (1 mentions)
10 protocols using aec chromogen
1
Immunohistochemistry of Skin Samples
2
Immunohistochemical Analysis of Extracellular Matrix
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Snap-frozen, optimal cutting temperature medium-embedded tissues were cut (7 µm) and fixed in 4% paraformaldehyde for 5 min, then incubated for 1 hour with TBS containing 2.5% normal goat serum+1% bovine serum albumin (BSA) and incubated 2 hours with goat anti-mouse Big-h3 (0.7 µg/mL in TBST 1% BSA, Santa Cruz), or rabbit anti-mouse vimentin (0.5 µg/mL in TBST 2% BSA, Cell Signaling) overnight at 4°C or rabbit anti-mouse fibronectin (0.5 µg/mL in TBST 2% BSA, Cell Signaling) 2 hours. After washing, endogenous peroxidase activity was blocked by incubating the slides with 0.3% H2O2 for 10 min. Secondary antibodies HRP-conjugated were then added for 1 hour in TBST 1% BSA. AEC chromogen (DAKO) was used and revelation reaction was stopped after 5 min, 10 min and 5 min, respectively. Counterstain with hematoxylin (Sigma) was performed. Slides were scanned using MIRAX digital microscope (Carl Zeiss MicroImaging), and images analyzed with MIRAX Viewer software (Zeiss).
3
Immunohistochemical Analysis of Breast Cancer
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Human tissue array (Cat No. BR1008) containing primary and lymph node metastastic breast cancer along with normal breast tissues was purchased from US Biomax, USA. The tissue array slides were deparaffinized with xylene twice and then rehydrated with distilled water through an ethanol series step by step. Tissue antigens were retrieved as described previously.47 (link) The slides were stained with polyclonal antibodies against KDM3A (1:100; Bethyl), JUN (1:200; Cell Signaling Technology), and MMP-9 (1:100; Santa Cruz Biotechnology) or control IgG (Santa Cruz Biotechnology) at 4°C overnight. We then incubated the sections with HRP-labeled polymer for 60 min, detected the immunocomplexes with AEC+ chromogen (Dako EnVision System), and counterstained with hematoxylin QS. The intensity of immunostaining was scored as follows: 0, no staining; +, weak staining; ++, moderate staining; +++, strong staining. The Wilcoxon rank sum test was applied to test the significant differences in immunohistochemical staining intensity between different groups. The Pearson correlation coefficient of linear regression was used to determine the correlation between different proteins. All statistical analyses were performed with SPSS 17.0 software.
4
Immunohistochemical Analysis of Liver and Spleen Tissues
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Specimens of excised livers and spleens were paraffin embedded and sectioned at 4 µm thickness. For histological assessment, deparaffinized liver sections were stained with hematoxylin (Merck, Darmstadt, Germany) and eosin Y (Carl Roth, Karlsruhe, Germany). For immunohistochemistry, deparaffinized organ sections were boiler-treated in EnVision™ FLEX Target Retrieval Solution, High pH (DAKO, Glostrup, Denmark) or 10 mM sodium citrate buffer pH 6.0 (applies to the NKp46 antibody only) for 30 min, blocked with 10% goat or 2.5% horse serum, incubated with the primary antibody overnight at 4°C (CD68: ab125212; CD3: ab5690; NKp46: ab214468 – all Abcam, Cambridge, UK), treated with 3% hydrogen peroxide and incubated with a biotinylated anti-rabbit secondary antibody (Vector Laboratories, Burlingame, CA, USA) at a dilution of 1:500. Peroxidase activity was developed with an avidin-biotin-enzyme complex (ABC) (Vector Laboratories) and AEC+ chromogen (DAKO), nuclei counterstained with hematoxylin, and sections mounted with aqueous mounting media, Aquatex® (Merck). Mounted sections were visualized using a Scope A.1 microscope and photomicrographed with an AxioCam MRC camera (Carl Zeiss Microscopy, Jena Germany). For quantification of NK cells, positively stained cells in sections treated with the NKp46 antibody were manually counted and calculated per mm2 of liver tissue.
5
Immunohistochemical Analysis of Annexin A2, CD44, Ki-67, and CD31
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The paraffin sections were first autoclaved in citrate buffer (pH 6.0) at 121°C for 10 min. Then sections were treated with 3% H2O2 and incubated with anti-Annexin A2, anti-CD44, anti-Ki-67 (550609; BD Biosciences), or anti-CD31 (ab9498; Abcam) antibodies diluted in antibody diluent (Biocare Medical, Concord, CA, USA) for 4°C overnight. After washing by PBS, the sections were allowed to react with the HRP-conjugated secondary antibodies, incubated with AEC chromogen (Dako, Carpinteria, CA, USA), and then counterstained with hematoxylin.
6
Histological Analysis of Cartilage Extracellular Matrix
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Harvested pellets were fixed in 3.5% neutral buffered formalin (Fischer), embedded in paraffin, and sectioned at 3–5 μm. Sections were heat fixed at 56°C for 24 h. Prior to staining, the sections were deparaffinized and rehydrated.10 (link)Immunohistochemical staining for collagen type I (anti-rat: ab34710, dilution 1:1000; Abcam, Cambridge, UK), collagen type II (anti-rat; dilution 1:4000; Developmental Studies Hybridoma Bank) and aggrecan (anti-rat, dilution 1:100; Millipore) was performed using LSAB + System-HRP (Dako, Agilent, Santa Clara, USA) and AEC chromogen (Dako, Agilent, Santa Clara, USA) as described elsewhere.10 (link)Histological staining with Alcian blue (Carl Roth, Karlsruhe, Germany) and with haematoxylin and eosin (Carl Roth, Karlsruhe, Germany) was carried out as previously described.10 (link)
7
Immunohistochemical Analysis of Angiogenic Factors
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In order to examine the localization and expression of VEGFR-1/Flt-1, VEGFR-2/Flk-1, HIF-1α, and iNOS, immunohistochemical analyses were performed. Hearts were fixed with formalin, embedded with paraffin, and cut to obtain 5 μm sections. After incubation of sections for 10 min with 0.3% H2O2, a serum-free protein block (DAKO, Carpinteria, USA) was added for 10 min. Sections were then incubated with the primary antibodies against VEGFR-1/Flt-1 (1:200; Chemicon® International, Serological Company, EU), VEGFR-2/Flk-1 (1:100; Chemicon® International, Serological Company, EU), HIF-1α (1:100; Chemicon® International, Serological Company, EU), and iNOS (1:50; Calbiochem®, San Diego, CA, USA) for 1 h at room temperature (RT). Non-immune mouse serum was substituted for negative controls. Before adding the primary antibodies for HIF-1α, the slides were treated with monohydrated citrate buffer (pH 6.0, 0.01 M) in a water bath for 40 and 10 min, respectively, at 100°C for the antigen retrieval. After incubation for 10 min with a biotinylated secondary antibody, AEC chromogen (DAKO, Carpinteria, CA, USA) was used to develop the horseradish peroxidase (HRP)-streptavidin complex.
8
Immunohistochemical Analysis of Breast Cancer
9
IHC Analysis of Colon Cancer TMA
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TMAs were purchased from US Biomax. Colon Cancer Screen TMA (BC05118a) contains 50 colon adenocarcinoma specimens with matching adjacent normal tissues. For immunostaining, tissue antigens were retrieved as described previously40 (link). The slides were then stained with monoclonal antibodies against β-catenin (1:100; BD Biosciences), polyclonal antibodies against KDM3A (1:100; Bethyl), and polyclonal antibody against KDM3B (1:200; Millipore) at 4 °C overnight in a humid chamber (Sigma). We then incubated the sections with HRP-labelled polymer for 60 min at room temperature, detected the immunocomplexes with AEC+ chromogen (Dako EnVision System) and counterstained with hematoxylin QS (Sigma). The intensity of immunostaining was scored as follows: 0, no staining; +, weak staining; ++, moderate staining; +++, strong staining. The Wilcoxon signed rank test was used to explore the significant differences in IHC staining intensity between different groups. All statistical analyses were performed using the SPSS 17.0 software.
10
Immunohistochemical Analysis of Myocardial MMP-9
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Matrix metalloproteinase (MMP)-9 is a proximal biomarker for cardiac remodelling and angiogenesis. The expression of myocardial MMP-9 was examined with immunohistochemical analyses as previous reported 21 (link). Hearts were fixed with formalin, embedded with paraffin and cut to obtain 5 μm sections. After incubation of sections for 10 min. with 0.3% H2O2, a serum-free protein block (DAKO, Carpinteria, CA, USA) was added for 10 min. Before adding MMP-9 primary antibody, the slides were treated with monohydrated citrate buffer (pH 6.0, 0.01 M) in a water bath for 10 min. at 100°C for the antigen retrieval. Sections were then incubated with the monoclonal antibodies against MMP-9 (1:100; Calbiochem®, San Diego, CA, USA) for 1 hour at room temperature. Anti-MMP-9 recognizes both latent and active form. Non-immune mouse serum was substituted for negative controls. After incubation for 10 min. with a biotinylated secondary antibody, AEC chromogen (DAKO) was used to develop the horseradish peroxidase-streptavidin complex.
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