Paraffin-embedded LADC tissue samples and cancer adjacent tissues were cut into 4 μm thick sections and affixed to the slides. The tissue sections were deparaffinized in xylene and rehydrated in a graded series of ethanol solutions using standard procedures. The sections were subsequently submerged in EDTA (pH 8) and autoclaved at 121°C for 5 min to retrieve the antigenicity. After washing in TBS, endogenous peroxidase was blocked by incubation in 3% hydrogen peroxide solution in methanol for 10 min at room temperature. Then, incubation with MAP4 antibody (Proteintech, Rosemont, IL, US) diluted at 1 : 4000 in TBS containing 0.5% BSA was carried out at 4°C overnight followed by further washing with buffer to remove unbound antibody. The sections were incubated with Envision secondary antibody (DAKO, Santa Clara, CA, US) for 30 min at room temperature. Chromogen (DAB) (GeneTex, Irvine, CA, US) was added to visualize the reaction. The sample was then counterstained with commercial hematoxylin (Beyotime Biotechnology, Nantong, Jiangsu, China), dehydrated sequentially in alcohols and xylene, and mounted.
Envision secondary antibody
Manufactured by Agilent Technologies
Sourced in United States, Denmark, United Kingdom
The Envision secondary antibody is a reagent used in immunohistochemistry and immunocytochemistry applications. It is designed to detect and amplify the signal from primary antibodies bound to target antigens in tissue or cell samples. The Envision secondary antibody contains multiple enzyme molecules that can catalyze the conversion of a chromogenic substrate, resulting in a visible color change at the site of the target antigen.
Automatically generated - may contain errors
Lab products found in correlation
Chromogen dab, by GeneTex (2 mentions)
Target retrieval solution, by Agilent Technologies (2 mentions)
Mouse anti sdhb monoclonal antibody clone21a11ae7, by Abcam (2 mentions)
Rabbit anti pdgfra monoclonalantibody clone d13c6, by Cell Signaling Technology (2 mentions)
Rabbit anti kit polyclonal antibody, by Agilent Technologies (2 mentions)
Proteinase k, by Merck Group (2 mentions)
Apoptag fluorescein in situ apoptosis detection kit, by Merck Group (2 mentions)
3 3 diaminobenzidine (dab), by Beyotime (2 mentions)
Ab15580, by Abcam (2 mentions)
Ab16669, by Abcam (2 mentions)
Haematoxylin, by Beyotime (1 mentions)
Rabbit anti ki67 antibody, by Abcam (1 mentions)
Dab substrate kit, by Vector Laboratories (1 mentions)
Rabbit anti ebf1 polyclonal antibody, by Abcam (1 mentions)
Rabbit anti cd133, by Abcam (1 mentions)
Fluorescein isothiocyanate fitc dextran, by Merck Group (1 mentions)
Neutral buffered formalin, by Merck Group (1 mentions)
Alexa fluor 488 conjugated goat anti rabbit igg, by Thermo Fisher Scientific (1 mentions)
Rabbit anti cleaved caspase 3, by Cell Signaling Technology (1 mentions)
Normal rabbit igg, by Santa Cruz Biotechnology (1 mentions)
19 protocols using envision secondary antibody
1
Immunohistochemical Analysis of MAP4 in LADC
2
Immunohistochemical Analysis of SDHB, PDGFRA, and KIT
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Immunohistochemistry was performed on 4-μm-thick paraffin
embedded tissue sections using a mouse anti-SDHB monoclonal antibody (clone
21A11AE7; 1:200 dilution; Abcam, Cambridge, MA), a rabbit anti-PDGFRA monoclonal
antibody (clone D13C6; 1:300 dilution; Cell Signaling Technology, Danvers, MA),
and a rabbit anti-KIT polyclonal antibody (1:150 dilution; Dako, Carpinteria,
CA). Pressure cooker antigen retrieval in citrate buffer (pH 6.1; Dako Target
Retrieval Solution) was used for PDGFRA and SDHB. Dako Envision+ secondary
antibody was used. The sections were developed using
3,3’-diaminobenzidine as substrate and counterstained with Mayer’s
hematoxylin.
embedded tissue sections using a mouse anti-SDHB monoclonal antibody (clone
21A11AE7; 1:200 dilution; Abcam, Cambridge, MA), a rabbit anti-PDGFRA monoclonal
antibody (clone D13C6; 1:300 dilution; Cell Signaling Technology, Danvers, MA),
and a rabbit anti-KIT polyclonal antibody (1:150 dilution; Dako, Carpinteria,
CA). Pressure cooker antigen retrieval in citrate buffer (pH 6.1; Dako Target
Retrieval Solution) was used for PDGFRA and SDHB. Dako Envision+ secondary
antibody was used. The sections were developed using
3,3’-diaminobenzidine as substrate and counterstained with Mayer’s
hematoxylin.
3
Immunohistochemical Staining of MAP4 Protein
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Conventional paraffin-embedded samples were precooled, and LADC tissue samples and adjacent tissues were cut into 4 μm thick slices and pasted onto slides. The samples were treated by xylene dewaxing 3 times and ethanol gradient dehydration. The sections were immersed in EDTA (pH 8) and autoclaved at 121 °C for 5 min to restore antigenicity. After cooling to room temperature, the sample was sealed in 3% hydrogen peroxide methanol solution for 10 min to eliminate endogenous peroxidase activity. MAP4 antibodies (Proteintech, Rosemont, IL, US) diluted at 1:4000 were incubated overnight at 4 °C, followed by further washing with buffers to remove unbound antibodies. At room temperature, the slices were incubated with Envision secondary antibody (DAKO, Santa Clara, CA, USA) for 30 min. The sample was cleaned with phosphate buffer solution (PBS) 3 times. Chromogen (DAB) (GeneTex, Irvine, CA, US) was used for colour development. Haematoxylin (Beyotime Biotechnology, Nantong, Jiangsu, China) was used for tissue restaining. Finally, the sections were dehydrated in alcohol and xylene and sealed. The results were observed under an optical microscope, and the images were analysed. Immunohistochemical staining evaluation was performed as described previously (Kwon et al. 2017 (link)).
4
Immunohistochemical Analysis of B-Cell Markers
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Immunohistochemical studies were performed on 4 micron tissue sections. IgJ immunostaining was performed manually with sodium citrate buffer heat-induced epitope retrieval, a 1/100 dilution of rabbit anti-human IgJ monoclonal antibody (clone SP105, Abcam, Toronto, ON; Antibody registry ID AB_10902174), and Envision secondary antibody (Dako, Burlington, ON). Immunohistochemistry for IgA, IgG, IgM, CD10, CD34, TdT, and PAX5 was performed using standard clinical antibodies and automated protocols on a Ventana Benchmark XT automated immunostainer (Tucson, AZ, USA). Normal tonsil and bone marrow was used as positive controls to demonstrate appropriate immunostaining patterns for all antibodies. Negative controls involved omission of primary antibody and showed negligible background staining.
5
Evaluating Varlitinib's Anti-Proliferative Effect
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To determine the anti-proliferative effect of varlitinib using the CCA xenograft model, immunostaining of Ki67 was performed on paraffin-embedded nude mouse tumor tissues. Nude mouse tumor tissue sections were deparaffinized in xylene followed by rehydration in a series of different ethanol concentrations. Then, the antigen was retrieved using Tris-EDTA buffer, pH 8.8, in a pressure cooker, and 0.3% H2O2 was used to block endogenous peroxidase activity for 30 mins with shaking. Non-specific binding was blocked by 10% skim milk in phosphate-buffered saline (PBS) for 30 min. Sections were incubated with rabbit anti-Ki67 antibody (Abcam, Cambridge, UK), dilution 1:200, at 4°C overnight in a moisture chamber. Sections were then incubated with peroxidase-conjugated EnVision™ secondary antibody (Dako, Denmark) followed by washing three times with working PBS for 5 min. Next, the color was developed with 0.1% diaminobenzidine tetrahydrochloride solution for 5 mins followed by counterstaining with Mayer’s hematoxylin. Sections were observed under a light microscope (Nikon H600L, Nikon, Japan). Ki67-positive cells of each tumor section were counted in at least five x400 power fields.
6
Immunohistochemical Analysis of EBF1 and CD133
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MMNK1 (60,000 cells/well), ox-MMNK1-L (60,000 cells/well), CCA cell lines (30,000 cells/well) were placed on 48-well plates for overnight. The cells were fixed with 4% paraformaldehyde-containing PBS for 30 min at room temperature. After washing, 0.2% (v/v) Triton-X100 solution was added. The cells were washed with PBS and incubated for 30 min with PBS containing 0.3% (v/v) hydrogen peroxide for endogenous hydrogen peroxide activity blocking and non-specific binding was blocked by 3% (w/v) BSA in PBS for 30 min. Cells were incubated with 10 µg/ml of rabbit anti-EBF1 polyclonal antibody (Abcam, MA, USA) or 9 µg/ml of rabbit anti-CD133 (Abcam, MA, USA) at room temperature for overnight followed by peroxidase-conjugated Envision™ secondary antibody (DAKO, Glostrup, Denmark). The color was developed with DAB substrate kit (Vector Laboratories, Inc., CA, USA) and washed with distilled water. The stained cells were dehydrated with stepwise (5 min/step) increasing concentrations of ethanol (70%→80%→90%→100%) and air dried overnight. The stained cells were examined under an inverted microscope.
7
Fluorescent Visualization of Cell-Cell Junctions
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Recombinant delta-toxin and rabbit anti-delta-toxin antibody were prepared as described previously [13 (link)]. Fluorescein isothiocyanate (FITC)–dextran (average mol wt 3000–5000), GI254023X, 10% neutral buffered formalin, and hydrogen peroxide were purchased from Merck (Tokyo, Japan). Rabbit anti-N-terminal fragment of E-cadherin antibody and normal rabbit IgG as an isotype control were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Trypsin inhibitor (TI), 3,3′-diaminobenzidine, HistoVT One, and Hanks’ balanced salt solution (HBSS) were obtained from Nacalai Tesque (Kyoto, Japan). Rabbit anti-cleaved caspase-3 was purchased from Cell Signaling Technology (Tokyo, Japan). A peroxidase-labeled anti-rabbit EnVision™ secondary antibody was obtained from Dako (Cambridge, UK). Alexa Fluor 488-conjugated goat anti-rabbit IgG and 4′,6-diamidino-2-phenylindole (DAPI) were obtained from ThermoFisher (Tokyo, Japan). All other chemicals were of the highest grade available from commercial sources.
8
Immunohistochemical Analysis of SDHB, PDGFRA, and KIT
9
Immunohistochemical and TUNEL Staining of Tumor Samples
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At the time of sacrifice, tumors were harvested and fixed in 4% paraformaldehyde for 24 hours. Fixed samples were embedded in paraffin and cut into 4 µm sections. The sections were deparaffinized in xylene and rehydrated with graded alcohol and then placed in 3% H2O2 to block endogenous peroxidase activity. After being boiled in 1 mM EDTA (pH 9.0) for antigen retrieval and blocked with 5% goat serum, sections were incubated with antibodies against mouse CD3 (ab16669, 1:150, Abcam) and Ki67 (ab15580, 1 µg/ml, Abcam) overnight at 4°C. After washing with PBS, sections were incubated in EnVision secondary antibody (Dako) for 30 mins at room temperature. Immunoreactivity was visualized with DAB (Beyotime Biotechnology). The sections were counterstained with hematoxylin. Tumor, lung, heart, liver, spleen, and kidney were also collected for histological analysis using hematoxylin and eosin (H&E) staining.
For TUNEL staining, the deparaffinized and rehydrated tissue sections were incubated in 20 g/ml proteinase K (Millipore) for 15 mins at room temperature. After being washed in PBS, the sections were stained using the ApopTag Fluorescein in situ Apoptosis Detection Kit (Millipore) according to the manufacturer’s (Millipore) protocol. DAPI (1.0 µg/mL) was used for nuclear counterstain.
For TUNEL staining, the deparaffinized and rehydrated tissue sections were incubated in 20 g/ml proteinase K (Millipore) for 15 mins at room temperature. After being washed in PBS, the sections were stained using the ApopTag Fluorescein in situ Apoptosis Detection Kit (Millipore) according to the manufacturer’s (Millipore) protocol. DAPI (1.0 µg/mL) was used for nuclear counterstain.
10
Immunohistochemical Analysis of Apoptosis and Macrophages in Intestinal Tissue
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Sections (5 µm) of paraffin-embedded small intestinal tissue were sectioned and used for immunohistochemistry. Following de-parafinization and rehydration, tissue sections were treated with 1% hydrogen peroxide in methanol to block endogenous peroxidases. Subsequently, slides were treated using heat-induced antigen retrieval in 0.01 M citrate acid buffer (pH 6) followed by incubation with a rabbit polyclonal anti-active caspase-3 (CC3) antibody (AF835: R&D Systems). Visualization of caspase-3 positivity was via a peroxidase-labelled anti-rabbit EnVision secondary antibody (Dako) and 3,3′-diaminobenzidine followed by counterstaining with haematoxylin. For macrophage staining, an antibody against F4/80 antigen (ab6640: Abcam) was employed using biotinylated anti-rat (BA-9401) and avidin–biotin reagent (PK-6100; Vector Laboratories).
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