Four-micrometer sections from formalin-fixed paraffin-embedded (FFPE) tissues were deparaffinized in xylene and rehydrated through graded alcohol washes. Antigen retrieval was performed by autoclaving in 0.01M citrate buffer (pH 6.0) for 3 minutes, followed by immersion in 3% hydrogen peroxide in methanol for 10 minutes to block endogenous peroxidase activity. The sections were then blocked with normal sheep serum (DAKO, Hamburg, Germany) for 90 minutes at room temperature and then incubated with MELK polyclonal antibody (Sigma-Aldrich, St. Louis, MO, USA) diluted at 1:300 overnight at 4°C. Diaminobenzidine was used as a chromogen, followed by counterstaining with hematoxylin. Samples were considered MELK-positive when 10% or more of the cancer cells had cytoplasmic MELK staining. The expression of MELK was assessed independently by two experienced pathologists who were blind to the patients' clinical outcomes. There was a high level of consistency between the two pathologists, and in the few discrepant cases (<5%) a consensus was reached after joint review.
Normal sheep serum
Manufactured by Agilent Technologies
Sourced in Germany
Normal sheep serum is a biological product derived from the blood of healthy sheep. It is a complex mixture of proteins, including immunoglobulins, hormones, and other biomolecules. This serum can be used in various research and laboratory applications as a blocking agent, diluent, or supplement in cell culture media.
Automatically generated - may contain errors
4 protocols using normal sheep serum
1
Immunohistochemical Analysis of MELK Expression in FFPE Tissues
2
TNFRSF11B Expression in Cancer Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Four micron sections were extracted from formalin fixed 70 paraffin-embedded (FFPE) tissues, dewaxed in xylene, and washed with graded alcohol for rehydration. The antigen retrieval was carried out in 0.01M citrate buffer (pH 6.0) by autoclaving for 3 minutes, and then immersed in methanol with 3% hydrogen peroxide for 10 minutes to block endogenous peroxidase activity. Then the sections were blocked by normal sheep serum (DAKO, Germany) for 1 hours at room temperature and after that incubated with TNFRSF11B polyclonal antibody (Abcam, MA, USA, Cat No: ab73400) diluted at 1:1000 for the night at 4°C. We used diaminobenzidine as a chromogen and hematoxylin was used as redyeing agent. The Samples were considered TNFRSF11B- positive when TNFRSF11B staining was present in the cytoplasm of 10% or more cancer cells. TNFRSF11B expression was independently evaluated by two experienced pathologists who had no knowledge of the patient's clinical outcomes. The two pathologists had a high degree of agreement, with a small number of differential cases (<5%) reaching agreement after joint review.
3
Immunofluorescence Staining for Active β-Catenin
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The cells were fixed in 4% paraformaldehyde solution and 1% Triton solution was added to penetrate the cell membrane. Normal sheep serum ((DAKO, Germany) was then used to block the cells at room temperature for 30 minutes. After incubating with primary antibodies (Active-β-Catenin Antibody, Merck, Germany, Cat No: 05-665-25UG ,1:100) at 4°C for 12-16 hours, Primary antibody binding and nucleus were detected in the dark using fluorescence secondary antibody (1:100) and Hoechst staining kit, respectively. A laser scanning confocal microscope (Leica, Germany) was used to observe the cells. In each experiment, at least 200 cells were counted.
4
Quantification of Tau and Amyloid-β in Brain Tissue
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Paraffin-embedded tissue sections (2 µm) were prepared from brain tissues fixed in 4% PFA (n = 5), autoclaved (120°C for 15 minutes) for antigen retrieval, and then treated with 0.3% H 2 O 2 in phosphate-buffered saline (PBS) to inactivate endogenous peroxidase. Blocking was performed with normal sheep serum (DAKO, Santa Clara, CA, USA) for 30 minutes at room temperature. Primary antibody (phosphorylated tau: Rb mAb to Tau S404, abcam; used at a dilution of 1:500) or Amyloid β42 (Aβ42) Rabbit Polyclonal Antibody (SIG-39131 SIGNET; used at a dilution of 1:500) (Signet Research Inc., Englewood, NJ, USA) was incubated overnight at 4 . After washing with PBS, Simple Stain Mouse MAX-PO(R) (Nichirei Biosciences, Tokyo, Japan) was added dropwise and allowed to react at room temperature for 30 minutes. Color development was performed using 3,3'diaminobenzidine tetrahydrochloride (DAB) (FUJIFILM Wako Pure Chem. Co.). Five areas of the cerebral cortex (area 54.6×20.6 µm 2 ) were photographed with an optical microscope FSX100 (OLYMPUS, Tokyo, Japan), and the number of p-tau cells were counted and the areas of Aβ accumulation were analyzed using an image-manipulating software (NIH Image version 1.41).
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!