The paraffin-embedded tissue arrays (BRN801a and BC081120, US Biomax, Rockville, MD) were dewaxed in xylene for 10 min twice and rehydrated through a series of alcohol solutions (200 proof, Sigma-Aldrich, St. Louis, MO) (100% ethanol twice, 90% ethanol, 70% ethanol, 5 min each) to water. Antigen retrieval was done by boiling the slides for 15 min in a citrate buffer (Teknova, U.S) at pH 6.0. Endogenous peroxidase activity was blocked with 3% H2O2 in methanol for 10 min after returning to room temperature. The TMAs were subsequently blocked with 2% BSA and incubated with monoclonal antibody (1:75) overnight at 4°C followed by incubation with a secondary antibody conjugated to horseradish peroxidase. Immunodetection was performed using DAB solution (Vector Laboratories, Burlingame, CA). Hematoxylin counterstain was used to visualize nuclei. The protein expression level in each tissue section was assessed in non-necrotic areas of three separate microscopic fields of view under a magnification of 20× and was represented by the mean of the percentage of positive cells.
Alcohol solutions
Manufactured by Merck Group
Alcohol solutions are a series of laboratory products provided by Merck Group. These solutions contain a specific concentration of alcohol, typically ethanol or isopropanol, in a balanced aqueous mixture. The core function of these alcohol solutions is to serve as solvents, disinfectants, or general-purpose cleaning agents in a laboratory setting.
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3 protocols using alcohol solutions
1
Immunohistochemical Analysis of Tissue Microarrays
2
Immunohistochemical Analysis of Tenascin-C Expression
3
Immunohistochemical Analysis of Tenascin-C Expression
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Immunohistochemical
staining was performed using tissue microarray
samples. The paraffin-embedded tissue arrays with 1.5 mm core diameter
and 5 μm thickness were dewaxed in xylene for 10 min twice and
rehydrated through a series of alcohol solutions (200 proof, Sigma-Aldrich,
St. Louis, MO) (100% ethanol twice, 90% ethanol, and 70% ethanol,
5 min each) to water. Then, the slides were boiled for 15 min in citrate
buffer (Teknova, Hollister, CA) at pH 6.0 for antigen retrieval. After
returning to room temperature, endogenous peroxidase activity was
blocked with 3% H2O2 in methanol for 10 min.
The TMAs were then rinsed with water and PBS and subsequently blocked
with 2% BSA and incubated with rabbit anti-human Tenascin-C monoclonal
antibody (1:100 dilution, Abcam, Cambridge, MA) overnight at 4 °C
followed by incubation with a goat anti-rabbit IgG conjugated to horseradish
peroxidase (1:250 Abcam, Cambridge, MA). Immunodetection was performed
using DAB solution (Vector Laboratories, Burlingame, CA). Hematoxylin
counterstain was used to visualize nuclei. The TNC expression level
in each tissue section was assessed in non-necrotic areas of three
separate microscopic fields of view under a magnification of 200×
and was represented by the mean of the percentage of TNC+ cells. The results were confirmed by a pathologist.
staining was performed using tissue microarray
samples. The paraffin-embedded tissue arrays with 1.5 mm core diameter
and 5 μm thickness were dewaxed in xylene for 10 min twice and
rehydrated through a series of alcohol solutions (200 proof, Sigma-Aldrich,
St. Louis, MO) (100% ethanol twice, 90% ethanol, and 70% ethanol,
5 min each) to water. Then, the slides were boiled for 15 min in citrate
buffer (Teknova, Hollister, CA) at pH 6.0 for antigen retrieval. After
returning to room temperature, endogenous peroxidase activity was
blocked with 3% H2O2 in methanol for 10 min.
The TMAs were then rinsed with water and PBS and subsequently blocked
with 2% BSA and incubated with rabbit anti-human Tenascin-C monoclonal
antibody (1:100 dilution, Abcam, Cambridge, MA) overnight at 4 °C
followed by incubation with a goat anti-rabbit IgG conjugated to horseradish
peroxidase (1:250 Abcam, Cambridge, MA). Immunodetection was performed
using DAB solution (Vector Laboratories, Burlingame, CA). Hematoxylin
counterstain was used to visualize nuclei. The TNC expression level
in each tissue section was assessed in non-necrotic areas of three
separate microscopic fields of view under a magnification of 200×
and was represented by the mean of the percentage of TNC+ cells. The results were confirmed by a pathologist.
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