TA muscles were carefully dissected and cut cross sectionally through the widest part of the muscle tissue. One half of the TA was embedded cut side down in OCT before being frozen in a bath of isopentane submerged in liquid nitrogen vapour. After freezing, blocks were brought to −20 °C and 5 μm sections were cut using a Leica Cryostat and then subjected to immunohistochemical staining for nitrotyrosine as described previously [34 (link)]. Briefly, mounted sections of TA muscle were fixed with cold acetone, and endogenous peroxidases were inactivated with 3% H2O2 in Tris-buffered saline. Sections were pre-blocked with a biotin-avidin blocking kit (Vector Laboratories) and then incubated with the nitrotyrosine antibody (Merck Milipore; 1:200) overnight at 4 °C. Subsequent secondary antibody, biotinylated anti-rabbit immunoglobulin 1:100 (Dako) was added for 30 min, followed by horseradish peroxidase–conjugated streptavidin, diluted 1:500 (Dako), and incubated for 30 min in 3,3′-diaminobenzidine tetrahydrochloride (DAB) (Sigma-Aldrich). Images were captured on an Olympus Slide scanner VS120 (Olympus) and viewed in OlyVIA (Olympus, build 13771) and quantitated using a singular threshold setting in Fiji across all samples [35 (link)].
Biotinylated anti rabbit immunoglobulin
Manufactured by Agilent Technologies
Sourced in Denmark, Japan, Australia
Biotinylated anti-rabbit immunoglobulin is a laboratory reagent used in various immunoassay techniques. It serves as a detection molecule that can bind to rabbit-derived antibodies, allowing for their visualization or isolation.
Automatically generated - may contain errors
Lab products found in correlation
Nitrotyrosine antibody, by Merck Group (1 mentions)
Horseradish peroxidase conjugated streptavidin, by Agilent Technologies (1 mentions)
Avidin biotin blocking kit, by Vector Laboratories (1 mentions)
3 3 diaminobenzidine tetrahydrochloride dab, by Merck Group (1 mentions)
Cryostat, by Leica (1 mentions)
Olyvia, by Olympus (1 mentions)
Vs120 slide scanner, by Olympus (1 mentions)
Normal horse serum, by Thermo Fisher Scientific (1 mentions)
Sc 109, by Santa Cruz Biotechnology (1 mentions)
Hydrogen peroxide, by Merck Group (1 mentions)
Peroxidase conjugated avidin, by Agilent Technologies (1 mentions)
Eclipse 200, by Nikon (1 mentions)
Plectin, by Abcam (1 mentions)
Streptavidin hrp, by Agilent Technologies (1 mentions)
Diaminobenzidine dab h2o2, by Merck Group (1 mentions)
Haematoxylin, by Merck Group (1 mentions)
4 protocols using biotinylated anti rabbit immunoglobulin
1
Quantitative Immunohistochemistry of Nitrotyrosine
2
Immunohistochemical Quantification of NF-kβ in Gastric Tissue
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Tissue sections were deparaffinized with xylene and ethanol for 10 min. The antigen was then retrieved with citrate buffer pH 6.0 in microwave for 13 min. Slides were incubated with 3% hydrogen peroxide (Merck, Hohenbrunn, Germany) for 5 min and with 3% normal horse serum (Gibco, Carlsbad, CA, USA) for 20 min to block endogenous peroxidase activity and nonspecific binding, respectively. Subsequently, gastric sections were incubated in a humidified chamber at room temperature for 1 h with a polyclonal antibody against NF-kβ (sc109; Santa Cruz Biotechnology, Santa Cruz, CA, USA) at a dilution of 1:100. Slides were then incubated with biotinylated anti-rabbit immunoglobulin (DAKO, Glostrup, Denmark) for 30 min. When the color development with diaminobenzidine (DAKO, Glostrup, Denmark) was seen, sections were counterstained with hematoxylin. Under light microscopy, positive cells were gastric epithelial cells with dark brown-stained nuclei. Under medium magnification (× 10 lens), one thousand gastric epithelial cells were manually counted for each rat to quantify the percentage of immune-positive cells. This procedure was performed by a pathologist who was blinded to the experiment. The percentage of immunoreactive cells was calculated as follows.
3
Immunohistochemical Analysis of Lymphatic Vessels
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
An immunohistochemical analysis of LYVE1 was performed using the avidin-biotinperoxidase complex method. Dewaxed and rehydrated mouse tumor tissue sections
were incubated overnight at 4°C with a rabbit monoclonal anti-mouse LYVE1 antibody at a 1:100 dilution or with PBS for negative control, then washed with PBS.
Biotinylated anti-rabbit immunoglobulin (DAKO, Kyoto, Japan) was then added to the sections for 30 minutes at room temperature. Peroxidase-conjugated avidin (DAKO, Kyoto, Japan) was then applied after the sections were washed with PBS.
The peroxidase activity was detected by exposing the sections to a solution of 0.05% 3, 3-diaminobenzidine and 0.01% H2O2 in Tris-HCl buffer (3, 3-diaminobenzidine solution) for 10 minutes at room temperature. The sections were counterstained with hematoxylin.
The stained sections were then analyzed using standard light microscopy (Nikon, Eclipse 200). Under low magnification, the most vascularized intratumoral areas were chosen (hot spots). The number of immunostained lymphatic vessels found in 3 hot spot areas at 400× magnification were counted. The LMVD for each case was expressed by the mean value (total number of vessels in 3 hot spot microscopic fields/3).
were incubated overnight at 4°C with a rabbit monoclonal anti-mouse LYVE1 antibody at a 1:100 dilution or with PBS for negative control, then washed with PBS.
Biotinylated anti-rabbit immunoglobulin (DAKO, Kyoto, Japan) was then added to the sections for 30 minutes at room temperature. Peroxidase-conjugated avidin (DAKO, Kyoto, Japan) was then applied after the sections were washed with PBS.
The peroxidase activity was detected by exposing the sections to a solution of 0.05% 3, 3-diaminobenzidine and 0.01% H2O2 in Tris-HCl buffer (3, 3-diaminobenzidine solution) for 10 minutes at room temperature. The sections were counterstained with hematoxylin.
The stained sections were then analyzed using standard light microscopy (Nikon, Eclipse 200). Under low magnification, the most vascularized intratumoral areas were chosen (hot spots). The number of immunostained lymphatic vessels found in 3 hot spot areas at 400× magnification were counted. The LMVD for each case was expressed by the mean value (total number of vessels in 3 hot spot microscopic fields/3).
4
Immunohistochemical Analysis of Plectin and α-Actin-2
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For the analysis of plectin and α-Actin-2 by IHC, 6 µm TMA sections were analysed as previously described [28] . Briefly, the tissue sections were dewaxed, rehydrated with xylene and ethanol and subjected to microwave antigen retrieval for 10 minutes at 100⁰C (Sixth Sense, Whirlpool, VIC, Australia) in 10mM citric acid buffer pH=6. TMA sections were incubated overnight at 4⁰C with either α-Actin-2 (1/500, rabbit polyclonal, ProteinTech, Chicago, USA) or plectin (1/250, rabbit monoclonal, Abcam, MA, USA) in blocking buffer (5% goat serum), followed by incubation with biotinylated antirabbit immunoglobulin (1/400, Dako, NSW, Australia) and streptavidin-HRP (1/500, Dako).
Immunoreactivity was detected using diaminobenzidine (DAB)/H2O2 (Sigma Aldrich) substrate and counterstaining with haematoxylin (Sigma Aldrich). TMA slides were digitally scanned using a Nanozoomer and images were obtained using NDP view imaging software. Analysis was carried out in IHC Profiler-Image J [29] . For each tissue core, three representative photo-micrographic images at 40x magnification were analysed.
Immunoreactivity was detected using diaminobenzidine (DAB)/H2O2 (Sigma Aldrich) substrate and counterstaining with haematoxylin (Sigma Aldrich). TMA slides were digitally scanned using a Nanozoomer and images were obtained using NDP view imaging software. Analysis was carried out in IHC Profiler-Image J [29] . For each tissue core, three representative photo-micrographic images at 40x magnification were analysed.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!