Paraffin-embedded sections (5 μm thick) from 10 patients were dewaxed, rehydrated, and treated with a citrate buffer (0.01 M, pH 6) to retrieve epitope. H2O2 was then used to block endogenous peroxidase activity. Immunodetection was carried out using an anti-OB-R antibody (1 : 200 dilution) (Cell Signaling, Danvers), and, after washing, incubation with a biotinylated goat anti-polyvalent antibody (Detection IHC kit, Abcam) was performed. Finally, staining was visualized using 3,3-diaminobenzidine tetrahydrochloride (Detection IHC kit, Abcam). The sections were slightly counterstained with Mayer hematoxylin (Carlo Erba Reagents S.r.l., Milan, Italy) and were analyzed using a binocular microscope with a 20x objective, a digital image capture computer system, and the software supplied with the microscope.
Ihc detection kit
Manufactured by Abcam
Sourced in United States, United Kingdom
The IHC Detection Kit is a laboratory tool designed for the detection of target proteins in tissue sections through immunohistochemistry (IHC) techniques. It provides the necessary reagents and components to facilitate the visualization of specific proteins of interest in biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Ab15580, by Abcam (1 mentions)
Ab187157, by Abcam (1 mentions)
Ab138386, by Abcam (1 mentions)
Hematoxylin, by Merck Group (1 mentions)
Aperio scanner system, by Leica (1 mentions)
Nanozoomer s60 digital slide scanner, by Hamamatsu Photonics (1 mentions)
St5020 cv5030 multistainer coverslipper, by Leica (1 mentions)
Ab131442, by Abcam (1 mentions)
Ab16663, by Abcam (1 mentions)
Ab109520, by Abcam (1 mentions)
Phospho histone h3 ser10, by Merck Group (1 mentions)
Cyclin d1, by Merck Group (1 mentions)
Cox 2, by Cell Signaling Technology (1 mentions)
Active β catenin, by Merck Group (1 mentions)
13 protocols using ihc detection kit
1
Immunodetection of OB-R Protein
2
Immunohistochemical Detection of Met and PTHR1
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From each paraffin block, tumor sections were sliced and prepared for immunohistochemical detection using the primary monoclonal antibody anti-Met (#8198, 1:2000) or anti-PTHR1 antibody (sc-12722-3D1.1, 1:50) according to the manufacturer’s protocol. The sections were deparaffinized with xylol for 15 min and subsequently rehydrated in decreasing concentrations of alcohol. Antigenic retrieval was carried out using a pressure cooker with sodium citrate buffer (10 mmol/L, pH 6) for 15 min at 1 atm. The sections were washed twice with PBS and we proceeded to block endogenous peroxidase with 30% H2O2for 10 min. After two washes with PBS, the corresponding antibody was added to each sample and incubated overnight at 4 °C. Immunohistochemical staining was carried out manually using ABCAM Detection IHC Kit (Abcam, Cambridge, MA, United States) according to the manufacturer’s instructions. The images obtained under an optical microscope were analyzed using an open-source image processing package based on ImageJ-NIH program[54 ,55 (link)].
3
Phospho-RSK Immunohistochemistry Protocol
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Paraffin-embedded sections were deparaffinized, re-hydrated and pre-treated using heat-mediated antigen retrieval (using a pressure cooker) with sodium citrate buffer (10 mM, pH 6) for 15 min. After the antigen retrieval step, sections were washed and blocked in 30% H 2 O 2 , and the primary antibody (anti-phospho RSK) was added for overnight incubations at 4°C. Immunohistochemical staining was carried out manually using ABCAM Detection IHC Kit (ABCAM) according to the manufacturer's instructions. Finally, the slides were monitored under microscope and the reaction was stopped with distilled water, counterstained with hematoxylin, dehydrated and coverslipped. Staining was visualized using a light microscope.
4
Immunohistochemical Analysis of Signaling Proteins
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Paraffin embedded sections were deparaffinised, re-hydrated and pre-treated using heat mediated antigen retrieval (using a pressure cooker) with sodium citrate buffer (10 mM, pH 6) for 15 min. After the antigen retrieval step, tissue sections were washed and blocked in 30% H 2 O 2 , and the primary antibodies (anti-phospho CREB/ATF-1, antiphospho ERK, anti-ERK 1/2, anti-Cyclin D1 and anti-phospho Akt) were added for overnight incubations at 4 °C. Immunohistochemical staining was carried out manually using ABCAM Detection IHC Kit (abcam, Cambridge, MA, USA) according to the manufacturer's instructions. Finally, the slides were monitored under microscope and the reaction was stopped with distilled water, counterstained with hematoxylin, dehydrated, and coverslipped. Staining was visualized using a light microscope.
5
Immunohistochemical and Viability Assays
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First, we deparaffinised the paraffin embedded sections and re-hydrated them.
Then, we performed the antigen retrieval using heat and a sodium citrate buffer (10 mM, pH 6) for 15 minutes. The sections were washed with PBS, blocked in 30% H 2 O 2 and incubated with the primary antibody (anti-VEGF or anti-CD31) overnight at 4ºC. We employed ABCAM Detection IHC Kit (ABCAM, Cambridge, MA, USA) according to the manufacturer's instructions. Finally, the reaction was stopped with distilled water according to microscopic observation and, counterstained with hematoxylin, dehydrated, and coverslipped. The slides were visualized using a light microscope. Endothelial cells were plated for triplicate in 96-well plates. After each treatment, 10 µl of the reagent was added on each well followed by 1 hour of incubation at 37 °C. Viable cells retain the ability to reduce resazurin, which is blue and nonfluorescent, into resorufin, which is red and brightly fluorescent. After the incubation step, the relative fluorescent units were measured.
Then, we performed the antigen retrieval using heat and a sodium citrate buffer (10 mM, pH 6) for 15 minutes. The sections were washed with PBS, blocked in 30% H 2 O 2 and incubated with the primary antibody (anti-VEGF or anti-CD31) overnight at 4ºC. We employed ABCAM Detection IHC Kit (ABCAM, Cambridge, MA, USA) according to the manufacturer's instructions. Finally, the reaction was stopped with distilled water according to microscopic observation and, counterstained with hematoxylin, dehydrated, and coverslipped. The slides were visualized using a light microscope. Endothelial cells were plated for triplicate in 96-well plates. After each treatment, 10 µl of the reagent was added on each well followed by 1 hour of incubation at 37 °C. Viable cells retain the ability to reduce resazurin, which is blue and nonfluorescent, into resorufin, which is red and brightly fluorescent. After the incubation step, the relative fluorescent units were measured.
6
Immunohistochemical Detection of Klebsiella pneumoniae
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Monospecific antisera produced by immunizing rabbits using hvKp isolates followed by absorption against non-hvKp were used. The tissue section that has been attached to a microscopic slide using poly-L-lysine 1% was deparaffinized, rehydrated with distilled water, and put under flowing with tap water for 5 min, followed with phosphate-buffered saline (PBS) Tween. The method followed the protocol that was supplied with the mouse and rabbit specific HRP/DAB (ABC) detection IHC kit (ab64264, Abcam, Cambridge, UK). Blocking endogenous activity was performed with immersion in 3% hydrogen peroxide at room temperature (20-25°C) for 15 min, followed by three washes in PBS Tween. Non-specific protein binding was blocked using 10% normal goat serum at 20-25°C for 30 min and washed with PBS Tween. Tissue sections were incubated overnight at 4°C with rabbit anti-K. pneumoniae polyclonal antibodies and followed by incubation with secondary antibody Dako REAL™ envision™/HRP, rabbit/mouse (ENV) for 30 min. The streptavidin-HRP was applied for 30 min at 20-25°C, and the section was visualized using Dako REAL™ DAB+chromogen in Dako REAL™ substrate buffer. The section was counterstained with Mayer’s hematoxylin and mounted with Aquamount.
7
Immunohistochemical Analysis of Tumor Samples
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Paraffin‐embedded tumor tissues were sliced and deparaffinized for gradient dehydration. Subsequently, placed in a staining jar containing EDTA, the slides underwent antigen retrieval performed by microwave heating. After dropping endogenous peroxidase blockers on the slides after cooling down, the slides were left at room temperature for 10 min. They were first incubated with primary antibodies, namely STK11 (1: 100, ab138386, Abcam), CD1E (1: 50, ab187157, Abcam), and Ki67 (1: 2000, ab15580, Abcam) for 1 h at 37°C and then for another 30 min with secondary antibodies at room temperature. 3,3′Diaminobenzidine (DAB) staining was performed using Rabbit Specific horseradish peroxidase/DAB (avidin‐biotin‐pcroxidase complex method) and Detection IHC Kit (ab64261, Abcam) according to the manual. After being counterstained with hematoxylin, the slides were mounted, followed by observing and photographing under the microscope.
8
Immunohistochemical Profiling of Cell-Cell Adhesion Markers
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Tissue microarray slides were purchased from Superchip Company (Shanghai, China). IHC was performed using the IHC Detection Kit (Abcam, ab64264) according to the manufacturer’s instructions. Paraffin-embedded tissue sections were deparaffinized, rehydrated, subjected to antigen retrieval, permeabilized, blocked, and incubated with primary antibodies against DSC2 (Invitrogen), PKP1 (Abcam), VIM (Cell Signaling Technology), and ZEB1 (Sigma-Aldrich) at 4°C overnight. Biotinylated secondary antibody was incubated for 30 min, and the antigen-antibody interaction was revealed with the streptavidin-biotin–horseradish peroxidase system using diaminobenzidine as a chromogen. Nuclei were counterstained with hematoxylin (Sigma-Aldrich). Images were obtained with an Aperio Scanner System (Leica) at a magnification of ×20. The expression levels of DSC2, PKP1, VIM, and ZEB1 were determined by semiquantitative analysis. IHC scores were graded according to percentage of stained cells as described (57 (link)). The percentage scores were assigned as follows: 0, none; 1, <1% of positively staining cells; 2, 1 to 10%; 3, 11 to 33%; 4, 34 to 66%; and 67 to 100%.
9
Immunohistochemical Detection of Toxoplasma gondii
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The tissues were processed using routine histological techniques and then embedded in paraffin. Paraffin sections (5 μm in thickness) of the samples were prepared and stained with hematoxylin and eosin (H&E). Immunohistochemistry (IHC) was conducted using rabbit anti-T. gondii serum as the primary antibody and mouse anti-rabbit IgG conjugated with HRP/DAB as the second antibody (IHC detection kit, Abcam, ab64264). Brain tissue sections of a VEG T. gondii-infected mouse were used as a positive control for IHC (kindly provided by Dr. Dubey, ARS, USDA).
10
Quantitative PAI-1 Expression Analysis in Tumors
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The catalogue numbers for the tissue microarray (TMA) slides were HMelC112CD01 for melanoma and F551101 for TNBC. Tumor samples were analysed using the IHC Detection Kit (ab64264, Abcam). AEC was used as a chromogen for melanoma samples, and diaminobenzidine was used for TNBC samples by strictly following the indicated protocols. Images were captured by the Aperio Scanner System. PAI-1 expression was scored manually based on staining intensity, ranging from 0 to 3 at an interval of 0.5. PAI-1 staining in each sample was scored as high (score ≥ 2), medium (1 ≤ score < 2) or low (score < 1) staining, and samples were classified as positive (score ≥ 1.5) or negative (score < 1.5) based on this score.
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