Cells were washed twice with ice-cold PBS and fixed in 4% paraformaldehyde in PBS for 20 min at 4°C. After washing 3 times with PBS, cells were permeabilized with 0.1% Triton X-100 for 5 min at 4°C and incubated with 1% BSA in PBS for 30 min, followed by incubation with primary antibody to p53 (1:1,000 dilution; product no. 2527, Cell Signaling Technology, Inc.) at 4°C overnight. The cells were then washed in PBS three times, followed by incubation with the secondary antibodies, FITC-conjugated goat anti-rabbit antibody (1:500 dilution; cat. no. 65-6111 Invitrogen; Thermo Fisher Scientific, Inc.) for 1 h at room temperature in the dark. Cells were washed with PBS and nuclei were stained with 0.5 µg/ml DAPI in PBS containing Tween-20 for 5 min. Samples were mounted with immunofluorescence mounting medium (Dako Cytomation; Agilent Technologies, Inc.) and images were captured under a fluorescence microscope using magnification of ×20.
Dakocytomation
Manufactured by Agilent Technologies
Sourced in United States
DakoCytomation is a line of laboratory equipment and instrumentation produced by Agilent Technologies. The core function of DakoCytomation products is to facilitate the analysis and processing of biological samples, including cells and tissues, for various research and diagnostic applications. DakoCytomation offers a range of specialized instruments and reagents designed to support immunohistochemistry, flow cytometry, and other analytical techniques.
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Lab products found in correlation
Ecl prime western blotting detection reagent, by GE Healthcare (1 mentions)
Imagequant las 4000, by GE Healthcare (1 mentions)
Anti gapdh antibody, by Merck Group (1 mentions)
Vectashield, by Vector Laboratories (1 mentions)
Eclipse 90i microscope, by Nikon (1 mentions)
Tissue tek, by Sakura Finetek (1 mentions)
Rabbit anti calretinin, by Swant (1 mentions)
Fluorescent mounting media, by Agilent Technologies (1 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 647, by Thermo Fisher Scientific (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions)
7 protocols using dakocytomation
1
Immunofluorescence Assay for p53 Protein
2
Immunohistochemical Analysis of Rat Stomach
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The immunohistochemical analysis of rat’s stomach tissue was conducted using DakoCytomation staining protocol (Agilent Technologies, Santa Clara, CA, USA).21 (link) In brief, the rat’s stomach tissues were cut at 5 µm thickness prior to deparaffinization and dehydration. Each section was then fixed on a glass slide treated with 3-aminopropyltrimethoxysilane and washed with a washing buffer. Each section of stomach was incubated for 15 min with a primary antibody (biotinylated), followed by staining with Hsp70 and Bax using streptavidin peroxidase detection reagents (Abcam, Cambridge, UK). A light microscope was used to observe the brown-stained regions which indicated positive findings.
3
Measuring Hepatic Stellate Cell Activation
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Alpha-smooth muscle actin (α-SMA) expression was examined by Western blot analysis because it was a major indicator of hepatic stellate cells (HSCs) activation which induced liver fibrosis [1 (link),2 (link),36 (link)]. Ten milligrams of proteins were subjected to Western blot analysis as previously described [29 (link),30 (link)]. Mouse monoclonal antibodies including anti-αSMA (DakoCytomation, Agilent Technologies, Inc., Santa Clara, CA, USA) and anti-GAPDH antibody (MilliporeSigma, Burlington, MA, USA) were used. Chemiluminescent images were acquired by ImageQuant LAS-4000 (GE Healthcare UK Ltd.) with ECL Prime Western Blotting Detection Reagent (GE Healthcare UK Ltd.).
4
Cryoprotection and Immunohistochemical Analysis
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A second set of specimens of wounds was cryoprotected using Tissue-Tek (Sakura Finetek Europe B.V.) and frozen in liquid nitrogen. Tissue sections (~10 µm thickness) were first mounted on the surface of poly-l-lysine-treated glass slides (Sigma-Aldrich; Merck KGaA), then fixed at room temperature using 2% (w/v) paraformaldehyde in PBS for 10 min. Non-specific binding of the secondary antibody was blocked at room temperature with normal swine serum (DakoCytomation; Agilent Technologies, Inc.) diluted with PBS (1:30) for 30 min.
Solutions of commercially available primary and secondary antibodies are shown inTable I . Incubation with primary and secondary antibodies was performed for 90 and 45 min (at room temperature), respectively. Nuclear DNA was stained using DAPI for 1 min at room temperature. Controls for specificity of the immunohistochemical detection were as follows: i) Replacement of the target-specific antibody by an irrelevant antibody (in the case of monoclonal antibodies of the same isotype); and ii) omission of the incubation step with the primary antibody to exclude antigen-independent signal generation. Specimens were mounted using Vectashield (Vector Laboratories, Inc.) and examined under an Eclipse 90i microscope (Nikon Corporation), as aforementioned.
Solutions of commercially available primary and secondary antibodies are shown in
5
Immunohistochemical Analysis of Hsp70 and Bax
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The immunohistochemical technique was conducted using (Dako cytomation, USA). Briefly, the tissue section slides were placed in a hot-air oven for 25 min at 60 °C (Venticell, MMM, Einrichtungen, Germany). De-paraffinization of the tissue sections was done using xylene and graded alcohol. After that, slides were boiled in antigen retrieval solution and then incubated with biotinylated primary antibodies of heat shock protein 70 (Hsp70) (1:500) and Bcl-2-associated protein x (Bax) (1:200) for 15 min. Subsequently, streptavidin conjugated to horseradish peroxidase was added to the slides and then incubated for 15 min. Further incubation for 5 min was done after adding DAB-substrate-chromagen to the slides. Finally, slides were immersed in hematoxylin for 5 sec, washed with distilled water and dipped in weak ammonia (0.037 mol/L) 10 times. Positive results of the immunohistochemical staining can be observed as brown areas under a light microscope.
6
Immunofluorescence Analysis of Axon Terminal Sprouting
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Four days (96 h) after rhodamine injection, deeply anesthetized animals (sodium pentobarbital, 100 mg/kg, i.p.) were perfused transcardially with 0.9% saline, followed by 4% paraformaldehyde in phosphate buffer 0.1 M, pH 7.4. Their brains were then dissected and cryoprotected in 30% sucrose prepared in phosphate buffered saline (PBS). Then, 40-µm-thick brainstem coronal sections were cut using a cryostat and collected in glycerol-PBS (1:1) for storage at −20 °C.
For the study of the effect of partial deafferentation and BDNF administration on axon terminal sprouting from abducens internuclear neurons, we used immunofluorescence against calretinin. Briefly, sections were rinsed, blocked with 7% normal goat serum (NGS) in PBS with 0.1% Triton X-100 (PBS-T), and incubated in the primary antibody (rabbit anti-calretinin, 1:5000; Swant, Burgdorf, Switzerland) at room temperature for 12 h. Sections were then washed and incubated for 2 h in the secondary antibody solution (goat anti-rabbit IgG coupled to Cy5, 1:200 in PBS-T; Jackson Immunoresearch, West Grove, PA, USA). After several washes in PBS, sections were mounted on glass slides and coverslipped with DakoCytomation (Dako, Glostrup, Denmark).
For the study of the effect of partial deafferentation and BDNF administration on axon terminal sprouting from abducens internuclear neurons, we used immunofluorescence against calretinin. Briefly, sections were rinsed, blocked with 7% normal goat serum (NGS) in PBS with 0.1% Triton X-100 (PBS-T), and incubated in the primary antibody (rabbit anti-calretinin, 1:5000; Swant, Burgdorf, Switzerland) at room temperature for 12 h. Sections were then washed and incubated for 2 h in the secondary antibody solution (goat anti-rabbit IgG coupled to Cy5, 1:200 in PBS-T; Jackson Immunoresearch, West Grove, PA, USA). After several washes in PBS, sections were mounted on glass slides and coverslipped with DakoCytomation (Dako, Glostrup, Denmark).
7
Immunofluorescent Staining of Tumor Vasculature
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Sections from the same tumors and samples from normal oral mucosa biopsies used for LCM were deparaffinized and rehydrated, and incubated in low pH antigen-retrieval solution (Dakocytomation; Dako, Carpinteria, CA) for 20 minutes at 95°C. Double staining was performed using antibodies against CD31 (Clone JC70A, Dako, Carpinteria, CA) and Grp78 (clone H-129, SantaCruz Biotechnology, Santa Cruz, CA). Alexa Fluor 488 or Alexa Fluor 647 (Molecular Probes, Eugene, OR) conjugated sencondary antibodies were used. Slides were counterstained with 3.5 µM DAPI (Sigma-Aldrich, St. Louis, MO), and mounted with Fluorescent Mounting Media (Dako, Carpinteria, CA). Images were taken with Olympus IX70 microscope (Tokyo, Japan) and analyzed by ImageJ software (National Institute of Health, Bethesda, MD, USA). Only CD31 positive cells lining tubular structures were considered blood vessels. Tissue samples were obtained from the University of Michigan School of Dentistry tissue core [19] (link).
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